Protective monoclonal antibodies targeting Nipah virus G protein and uses thereof

Abstract

The present invention provides protective monoclonal antibodies targeting Nipah virus (NiV) G protein and their uses, wherein the monoclonal antibodies can recognize Nipah virus G protein. The present invention uses NiV G protein as an antigen target, displays antigens on a ferritin nanoparticle platform to immunize mice, and screens out two monoclonal antibodies LN1F9 and LN1D11 that can specifically bind to NiV G protein. In vitro neutralization experiments have shown that these two antibodies have high neutralizing activity against both NiV-M and NiV-B strains, among which LN1F9 can also neutralize Hendra virus (HeV) of the same genus. The monoclonal antibodies LN1F9 and LN1D11 can effectively treat hamsters infected with NiV and prevent NiV infection in hamsters. They have extremely high application value in clinical treatment and prevention of NiV and HeV infection, and can be used to prepare detection products and preventive and therapeutic drugs for NiV and HeV.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a protective monoclonal antibody targeting Nipah virus G protein and a use thereof. Background Art

[0002] Nipah virus (NiV) is a highly contagious, single-stranded, negative-sense RNA virus that infects humans and animals. It belongs to the genus Henipavirus (HNV) in the family Paramyxoviridae. Human infection with NiV can cause asymptomatic or mild influenza-like illness, while severe cases can develop fatal respiratory or neurological diseases, with a mortality rate as high as 70%. There are two main strains of NiV: the Malaysian strain (NiV-M) and the Bangladeshi strain (NiV-B). Over the past 20 years, NiV outbreaks have occurred almost annually in Bangladesh and India. In recent years, the range of HNV infections has continued to expand. In addition to the previously discovered Hendra virus (HeV), new viruses have been discovered in Africa, Australia, Asia, and South America. The newly discovered Mòjiāngvirus (MojV) and Langyavirus (LayV) in my country are also HNVs. Currently, there are no approved vaccines or antiviral treatments for NiV.

[0003] The World Health Organization and the U.S. Centers for Disease Control and Prevention have repeatedly designated NiV as a pathogen requiring high-priority attention. The development of candidate antiviral drugs is crucial for preventing and controlling NiV outbreaks, which pose a potential pandemic risk. NiV has two membrane-anchored glycoproteins, attachment protein G and fusion protein F, which work together to mediate viral entry into host cells. NiV's G and F proteins, located on the surface of the virion, play a crucial role in NiV infection. They are the primary structural proteins that induce the production of neutralizing antibodies and are important targets for NiV vaccine development. Studies have shown that antibodies targeting HeV's F and G proteins can neutralize and inhibit HeV infection, and some of these antibodies can even cross-recognize NiV.

[0004] Although some antibodies targeting the G protein head domain have been screened from recovered HeV patients, there is still a lack of approved antibody drugs against HNVs. This suggests that there is still a need to develop a group of protective antibodies with broad-spectrum neutralizing activity to provide new drug candidates and strategies for the prevention and control of HNV infections and lay the foundation for the construction of antibody cocktails against NiV and other HNVs. Summary of the Invention

[0005] In order to solve the technical problem, the present invention aims to provide two protective monoclonal antibodies targeting Nipah virus G protein and their uses. These two antibodies can specifically recognize two epitopes of Nipah virus G protein and have high in vitro neutralizing activity.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect of the present invention, protective monoclonal antibodies targeting Nipah virus G protein are provided, wherein the two protective monoclonal antibodies targeting Nipah virus G protein or antigen-binding fragments thereof each comprise a heavy chain variable region and a light chain variable region, and include one of the following monoclonal antibodies:

[0008] Monoclonal antibody LN1F9 or an antigen-binding fragment thereof: capable of recognizing the G protein of Nipah virus and Hendra virus, wherein the heavy chain variable region thereof has three complementarity determining regions of the amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 3; and the light chain variable region thereof has three complementarity determining regions of the amino acid sequence set forth in SEQ ID NO: 6 to SEQ ID NO: 8;

[0009] Monoclonal antibody LN1D11 or its antigen-binding fragment: capable of recognizing the G protein of Nipah virus, its heavy chain variable region has three complementarity determining regions of the amino acid sequence shown in SEQ ID NO: 15 to SEQ ID NO: 17; its light chain variable region has three complementarity determining regions of the amino acid sequence shown in SEQ ID NO: 20 to SEQ ID NO: 22.

[0010] Wherein, the amino acid sequence of the above SEQ ID NO: 7 is: NAK.

[0011] The amino acid sequence of the above SEQ ID NO: 21 is: YAS.

[0012] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody LN1F9 is shown in SEQ ID NO: 4; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 9;

[0013] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody LN1D11 is shown in SEQ ID NO: 18; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 23.

[0014] Furthermore, the monoclonal antibody LN1F9 contains a heavy chain as shown in SEQ ID NO: 5, and a light chain as shown in SEQ ID NO: 10.

[0015] Furthermore, the monoclonal antibody LN1D11 contains a heavy chain as shown in SEQ ID NO: 19, and a light chain as shown in SEQ ID NO: 24.

[0016] Furthermore, the monoclonal antibody further comprises:

[0017] Fab, Fab', Fab'-SH, scFv, F(ab')2 with the same antigen-binding fragment;

[0018] Or an antibody having the same function obtained by substituting, deleting and / or adding one or more amino acids to the amino acid sequence of the monoclonal antibody, comprising a heavy chain variable region having an amino acid sequence that is at least 80% homologous to the heavy chain variable region; and a light chain variable region having an amino acid sequence that is at least 80% homologous to the light chain variable region;

[0019] Or an antibody obtained by connecting a tag to the N-terminus and / or C-terminus of the monoclonal antibody;

[0020] Alternatively, the monoclonal antibody may be humanized to obtain an antibody with the same or similar function.

[0021] In other embodiments, V H and / or V L The amino acid sequence may be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the above sequence. H and V L V H and V L Antibodies to the regions can be obtained by mutagenizing (e.g., site-directed mutagenesis, PCR-mediated mutagenesis, and AI-mediated antibody engineering) nucleic acid molecules encoding SEQ ID NOs: 1-10 and 15-24, and then testing the encoded altered antibodies for retained function using the functional assays described herein.

[0022] The monoclonal antibodies include: mouse antibodies, humanized antibodies, bi / tri-specific antibodies or chimeric antibodies.

[0023] In other embodiments, the variable region gene can be converted into a scFv gene. Once the V encoding H and V L The fragmented DNA fragments can be further manipulated by standard recombinant DNA technology, for example, the variable region gene is converted into a full-length antibody chain gene, a Fab fragment gene or a scFv gene.

[0024] In these operations, the encoding V L or V HThe DNA fragment of the present invention is operably linked to another DNA fragment encoding another protein such as an antibody constant region or a flexible linker. As used herein, the term "operably linked" means that the two DNA fragments are linked together so that the amino acid sequences encoded by the two DNA fragments remain in the reading frame.

[0025] In the second aspect of the present invention, a polypeptide containing the amino acid sequence is provided.

[0026] Furthermore, the polypeptide contains an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 23, and SEQ ID NO: 24.

[0027] In the third aspect of the present invention, a nucleic acid molecule encoding the monoclonal antibody is provided, wherein the nucleic acid molecule comprises a nucleic acid molecule encoding the heavy chain variable region and a nucleic acid molecule encoding the light chain variable region.

[0028] The nucleic acid molecule encodes any of the aforementioned monoclonal antibodies or antigen-binding fragments thereof or polypeptides, including sequences encoding the heavy chain variable region (e.g., SEQ ID NO: 11) and / or the light chain variable region (e.g., SEQ ID NO: 13) of the LN1F9 antibody, as well as sequences encoding the heavy chain (e.g., SEQ ID NO: 12) and / or the light chain (e.g., SEQ ID NO: 14) of the LN1F9 antibody.

[0029] These include sequences encoding the heavy chain variable region (such as SEQ ID NO: 25) and / or light chain variable region (such as SEQ ID NO: 27) of the LN1D11 antibody, as well as sequences encoding the heavy chain (such as SEQ ID NO: 26) and / or light chain (such as SEQ ID NO: 28) of the LN1D11 antibody.

[0030] In a fourth aspect of the present invention, an expression vector comprising the nucleic acid is provided, wherein the expression vector is capable of expressing the nucleic acid in a prokaryotic or eukaryotic host cell.

[0031] The expression vector may specifically be, but is not limited to, a prokaryotic expression vector, a phage vector, a viral vector or a mammalian expression vector. The present invention specifically uses a mammalian expression vector.

[0032] In the fifth aspect of the present invention, an engineered bacterium or eukaryotic host cell comprising the expression vector is provided.

[0033] In the sixth aspect of the present invention, provided is the use of the protective monoclonal antibody targeting Nipah virus G protein, or the polypeptide, or the nucleic acid molecule, or the expression vector, or the engineered bacteria, or the eukaryotic host cell in the preparation of a drug for preventing and treating Nipah virus infection or a reagent for detecting Nipah virus.

[0034] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0035] Among the protective monoclonal antibodies targeting Nipah virus G protein provided by the present invention, two monoclonal antibodies LN1F9 and LN1D11 with high neutralizing activity binding to NiV G protein were obtained by screening NiV G-specific plasma cells of mice;

[0036] The monoclonal antibodies screened had high in vitro neutralizing activity, among which LN1F9 had an IC of 0.05 for NiV-M pseudovirus with VSV as the backbone. 50 The neutralization IC value for VSV-NiV-B pseudovirus can be as low as 0.0719 ng / mL 50 As low as 0.108ng / mL; the neutralization IC of LN1D11 against NiV-M pseudovirus with VSV as the backbone 50 The neutralization IC value for VSV-NiV-B pseudovirus can be as low as 0.657 ng / mL 50 The two NiV G-specific monoclonal antibodies of the present invention have relatively high application value in the clinical treatment and prevention of NiV and HeV infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following is a brief introduction to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0038] Figure 1Figures 1 and 2 show Coomassie Brilliant Blue staining and molecular sieve chromatograms of NiV G-ferritin nanoparticle immunogen SDS-PAGE electrophoresis. A shows the Coomassie Brilliant Blue staining of NiV G-ferritin nanoparticle immunogen under reducing (+) and non-reducing (-) conditions. B shows the Coomassie Brilliant Blue staining of NiV G head domain (NiV sG) under reducing (+) and non-reducing (-) conditions. C shows the Coomassie Brilliant Blue staining of ferritin under reducing (+) and non-reducing (-) conditions. D shows the molecular sieve chromatogram of NiV G-ferritin nanoparticles. E shows the molecular sieve chromatogram of NiV sG. F shows the molecular sieve chromatogram of ferritin.

[0039] Figure 2 For the mouse immunization process and flow cytometry sorting frame gate strategy; Figure 2 A is the immune operation process, Figure 2 B is the flow cytometry sorting result, where the red box represents the final sorted cells.

[0040] Figure 3 To amplify antibody variable region genes from single B cells; wherein, Figure 3 A is a nucleic acid gel image of the second round of heavy chain variable region gene cloning in the first 96-well plate of NiV G-specific single B cells after flow cytometry sorting. Figure 3 B is a nucleic acid gel image of the second round of light chain variable region gene cloning in the first 96-well plate of NiV G-specific single B cells after flow cytometry sorting.

[0041] Figure 4 The gel images of the purification of LN1F9 and LN1D11 antibodies under reducing (+) and non-reducing conditions (-); Figure 4 A is the SDS-PAGE image of LN1F9 antibody purification, Figure 4 B is the SDS-PAGE of LN1D11 antibody purification.

[0042] Figure 5 is the binding curve of two monoclonal antibodies to NiV G protein; Figure 5 A is the binding curve of LN1F9 antibody and NiV-MG protein, Figure 5 B is the binding curve of LN1D11 antibody and NiV-M G protein.

[0043] Figure 6 are the neutralization curves of the two monoclonal antibodies against VSV-NiV-M and VSV-NiV-B pseudoviruses respectively; Figure 6 A is the neutralization curve of LN1F9 monoclonal antibody against VSV-NiV-M pseudovirus, Figure 6B is the neutralization curve of LN1F9 monoclonal antibody against VSV-NiV-B pseudovirus, Figure 6 C is the neutralization curve of LN1F9 monoclonal antibody against VSV-HeV pseudovirus, Figure 6 Figure D is the neutralization curve of LN1D11 monoclonal antibody against VSV-NiV-M pseudovirus, and Figure E is the neutralization curve of LN1D11 monoclonal antibody against VSV-NiV-B pseudovirus.

[0044] Figure 7 The results of two monoclonal antibodies neutralizing live viruses; among them, Figure 7 A is the half-maximal inhibitory concentration (IC) of LN1F9 monoclonal antibody against live NiV-M, NiV-B and HeV viruses, respectively. 50 ), Figure 7 B is the IC of LN1D11 monoclonal antibody against live NiV-M and NiV-B viruses, respectively. 50 .

[0045] Figure 8 The figures are the protection results of monoclonal antibodies LN1F9 and LN1D11 in hamsters challenged with the virus. Figure 8 A shows the results of monoclonal antibodies LN1F9 and LN1D11 in preventing Syrian golden hamsters from NiV-M infection. Figure 8 B shows the results of monoclonal antibodies LN1F9 and LN1D11 in treating Syrian golden hamsters infected with NiV-M. DETAILED DESCRIPTION

[0046] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0047] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, 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 invention belongs. In the event of any conflict, the present specification shall take precedence.

[0048] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0049] The monoclonal antibodies of the present application, their preparation methods, and their application effects are described in detail below, with reference to the examples and experimental data. Specific experimental conditions and methods not specified in the following examples are generally based on conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual (3rd Edition), edited by J. Sambrook et al., Science Press, 1992; Cell Experiment Guide, edited by D.L. Spector et al., Science Press, 2001, or as recommended by the manufacturer.

[0050] Example 1 Construction, expression and purification of NiV G-ferritin nanoparticle immunogen

[0051] 1. NiV G-ferritin Nanoparticle Immunogen Gene Synthesis and Vector Construction

[0052] NiV G-ferritin consists of the following sequences from its N-terminus to its C-terminus: an N-terminal 6×His residue, the head domain of the Nipah virus Malaysian strain G protein (GenBanK: NP_112027.1) (hereinafter referred to as NiV G protein), and bullfrog-Helicobacter pylori ferritin. The NiV G protein head domain consists of positions 176-602 of the full-length G protein sequence. The bullfrog-Helicobacter pylori ferritin is derived by fusion of amino acids 2-9 of bullfrog ferritin (UniProt: P07797, containing an N8Q mutation, with the amino acid sequence ESQVRQQF) with the N-terminus of amino acids 3-167 of Helicobacter pylori ferritin (GenBanK: WP_000949190.1). The gene sequence was codon-optimized by GenScript (Nanjing, China) and synthesized by Qingke Biotechnology Co., Ltd. The synthesized gene fragment was constructed and expressed in the mammalian expression vector pFM1.2R (other commercially available eukaryotic expression vectors, such as the pCAGGGS vector (Cat. No. VT1076) from Ubao Biotechnology, can also be used). In addition to the NiV G-ferritin expression plasmid, expression plasmids for the NiV G protein head domain (containing a 6×His residue at the N-terminus, hereinafter referred to as NiV sG) and ferritin expression plasmid (containing a 6×His residue at the N-terminus) were also constructed. The expression vectors used for these two plasmids are the same as those used for the NiV G-ferritin expression plasmid.

[0053] The amino acid sequences of the target gene fragments inserted into the three plasmids, namely the NiV G-ferritin expression plasmid, the NiV G protein head domain expression plasmid (referred to as NiV sG expression vector) and the ferritin expression plasmid, are shown in SEQ ID NO.43 to SEQ ID NO.45.

[0054] 2. Protein Expression and Purification

[0055] NiV G-ferritin expression plasmid and NiV sG expression vector were transiently transfected into Expi293 cells using polyethyleneimine (PEI, Polyscience). The transfected cells were incubated at 100 rpm, 37 ° C and 8% CO2, and the supernatant was collected approximately 108 h after transfection. The supernatant was filtered through a 0.45 μm filter and purified with HighAffinity Ni-Charged Resin FF (GenScript). Soluble protein was further purified by size exclusion chromatography (SEC) using a Superose 6 Increase 10 / 300GL column (Cytiva) or a Superdex 200 Increase 10 / 300GL column (Cytiva) in 20 mM Tris and 150 mM NaCl pH 8.0. Purity was checked by SDS-PAGE.

[0056] 108 hours after transfection with the ferritin expression vector, the transfected cell pellet was harvested. The cell pellet was resuspended in 100 mL of lysis buffer (50 mM Tris pH 7.5, 100 mM NaCl, 0.25% NP-40, 100 μg / mL PMSF) and the cells were disrupted approximately 50 times using a Dounce grinder. Then, the cells were centrifuged at 12,000 × g for 40 min to remove cell debris. The supernatant was filtered through a 0.45 μm filter and purified with HighAffinity Ni-TED Resin FF (GenScript). The protein eluted with 500 mM and 1 M imidazole in 20 mM Tris and 150 mM NaCl pH 8.0 was purified by size exclusion chromatography using a Superose 6 Increase 10 / 300 GL column (Cytiva). Purity was checked by SDS-PAGE.

[0057] The purification results are as follows Figure 1 As shown in AC, SDS-PAGE results showed that the purified NiV G-ferritin, NiVsG and ferritin were of high purity, with sizes of approximately 70 kDa, 50 kDa and 19 kDa, respectively, which were close to the predicted monomer molecular weight. Figure 1 As shown in DF, the elution peak of NiV G-ferritin is earlier than that of ferritin and NiVsG, indicating that the nanoparticles are successfully assembled.

[0058] Example 2 Mouse immunization process and flow cytometry gate sorting strategy

[0059] 1. Mouse Immunization Process

[0060] Six-week-old BALB / c mice were purchased from Beijing Weitong Lihua. 1 μg or 10 μg of NiV G-ferritin nanoparticle vaccine was thoroughly mixed with an equal volume of AddaVax adjuvant and injected intramuscularly into each mouse for a total of three immunizations, with a three-week interval between immunizations. Blood was collected from the orbital venous plexus every week after the first immunization and every three weeks after the second and third immunizations, and serum antibody titers were measured. Twenty-one days after the third immunization, mice were euthanized, and spleens and lymph nodes were isolated and placed on a grinding mesh. The spleen or lymph nodes were gently ground using the rubber side of a 2 mL syringe. After grinding, the grinding mesh was rinsed with culture medium, and the suspension was collected. The mixture was centrifuged at 500 × g for 4 minutes, the supernatant was removed, and 3 mL of red blood cell lysis buffer was added. The mixture was incubated at room temperature for 3 minutes. The reaction was terminated by adding 7 mL of 1640 (2% FBS), mixed by inversion, and centrifuged again at 500 × g for 4 minutes, and the supernatant was removed. After resuspending in 500 μL 1640 (2% FBS), count the cells on a cell counting plate (usually mouse spleen cells are counted at 10 8 Schematic diagram of mouse immunization process is as follows Figure 2 As shown in A.

[0061] 2. Flow cytometry sorting gate strategy

[0062] 2.1 Flow staining steps:

[0063] (1) Prepare 8 single-stained tubes of cells (780APC-Cy7, CD3,4,8AmyCyan-A, CD19PE-Cy7, IgDPerCP-Cy5-5, CD138 FITC-A, CD95 PE-A, CD19-APC), 0.5×10 6 Cells / tube, 100 μL per tube (780 single-stained tube cells must undergo special treatment; 50 μL of cells are treated at 65°C for 1 minute and then combined with the original cells at a 1:1 ratio). Add the dye to the 96-well plate containing the single-stained tube cells according to the recommended antibody concentration in the manufacturer's instructions and incubate at 4°C in the dark for 30 minutes. Wash the cells twice with 200 μL of staining buffer and transfer the sample to a flow cytometer.

[0064] (2) The experimental group was the NiV G-ferritin nanoparticle vaccine group, and the control group was the ferritin group. The cells in the experimental and control groups were first incubated with 0.5 μg / mL randomly biotinylated NiV G head domain protein (EZ-Link NHS-PEG4-Biotin, ThermoFisher) at 4°C in the dark for 30 min, centrifuged, washed twice with 200 μL staining buffer, and then incubated with dye mixture (dye mixture was 780APC-Cy7, CD3,4,8AmyCyan-A, CD19 PE-Cy7, IgDPerCP-Cy5-5, CD138 FITC-A, CD95 PE-A, APC strep, diluted with staining buffer according to the antibody concentration recommended in the instructions). After washing the cells twice with 200 μL staining buffer, the samples were transferred to flow cytometry tubes.

[0065] 2.2 Flow sorting:

[0066] When loading the machine, first load the blank tube with cells, adjust the voltage, find the target cell population, then load the single-stained tube and adjust the compensation. Sorting target cell population - plasma cell PB: 780 - ,CD3 / 4 / 8 - ,CD19 + ,IgD - ,CD95 + and NiV G + .

[0067] The results of flow cytometry separation are as follows Figure 2 As shown in B, compared with the control group, the proportion of positive cell populations in the lymph node cells of the mice in the NiV G-ferritin group was significantly increased (0% vs 5.51%).

[0068] Single positive cells were sorted into a 96-well plate filled with lysis buffer (lysis buffer formula for one 96-well plate: 7.2 μL Tris + 18 μL Rnain (40 U / μL) + 694.8 μL Rnase-free water. 58 μL / well in the first row, and 7 μL was equally divided into each well with a dispenser) and stored in a -80°C refrigerator.

[0069] Example 3 Amplification of Antibody Variable Region Genes from Single B Cells

[0070] 1. Reverse transcription (using Novozymes R211 HiScriptII 1st Strand cDNA Sythesis Kit)

[0071] (1) After sorting, transfer the 96-well PCR plate containing 7 μL of catch buffer per well from -80°C to ice, let it stand for 5 min, and then centrifuge at 400 × g for 30 s at 4°C.

[0072] (2) Place the 96-well plate in a matching PCR instrument, incubate at 65°C for 5 minutes, and then quickly cool on ice for 2 minutes.

[0073] (3) Prepare the first-strand cDNA synthesis reaction using the following system: 10 μL of 2×RT Mix, 2 μL of HiScript III Enzyme Mix, and 1 μL of Random Hexamers per well. PCR protocol: reverse transcription at 50°C for 15 min, followed by reaction at 85°C for 2 min.

[0074] 2. First round of PCR

[0075] (1) Prepare the primer premix: Mix equal volumes of 1mFH_I to 1mFH_XI, 1mFK_I to 1mFK_X, or 1mFL_I to 1mFL_II forward primers and dilute to a concentration of 5 μM per primer. This is the forward primer premix. The first-round PCR primer sequences are shown in Table 1.

[0076] Table 1. First round PCR primers

[0077]

[0078]

[0079] (3) Prepare the first round of PCR reaction system: Prepare according to the instructions (Novozymes, P505-d3), including: 2× Phanta Buffer 10 μL, dNTP (10 mM) 0.4 μL, Phanta polymerase 0.4 μL, upstream primer mixture (5 μM each primer) 0.15 μL, downstream primer (50 μM) 0.1 μL, reverse transcribed cDNA 1.5 μL, and add double distilled water to make up to 20 μL.

[0080] The PCR program was set as follows: 95°C pre-denaturation for 3 min, followed by 50 cycles of 95°C denaturation for 15 s, 46°C annealing for 15 s, 72°C extension for 1 min, and a final extension at 72°C for 10 min. The product was used for the next round of PCR or stored at -20 / 80°C.

[0081] 3. Second round of PCR (seq-PCR, using 2× ES Taq PCR Mix, Comverse Century)

[0082] (1) Take 1.5 μL of the first-round PCR product as a template and perform seq-PCR. The amplification primers are shown in Table 2 below.

[0083] Table 2. Seq-PCR primers

[0084]

[0085] (2) The PCR reaction system was prepared according to the manufacturer's instructions (Kangwei Century, EsTaq), including: 6 μL of 2× EsTaq Mix, 0.1 μL of upstream primer (50 μM), 0.1 μL of downstream primer (50 μM), 1.5 μL of the first-round PCR product, and double-distilled water to 12 μL. The PCR program was set as follows: 94°C pre-denaturation for 2 min, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 20 s, with a final extension at 72°C for 10 min.

[0086] (3) Prepare a 1% agarose gel and run it to identify the size. The band should be between 400 and 600 bp. Figure 3 shown.

[0087] (4) Monoclonal PCR products with bands of the correct size for both IgH and IgK / L were sequenced using reverse primers. The sequencing results were verified using NCBI's BLAST or IMGT database to determine if the antibody sequence was available.

[0088] 4. The third round of PCR (cloning PCR, using high-fidelity enzyme, NoviZan Phanta-P505-d3)

[0089] (1) Prepare primer premix: Mix equal volumes of upstream primers mVH01-F to mVH23-F (50 μM stock, diluted to 1 μM each), upstream primers mVK01-F to mVK28-F (50 μM stock, diluted to 1 μM each), and downstream primers mJH01-R to mJH04-R and mJK01-R to mJK05-R (50 μM stock, diluted to 5 μM each). Primer sequences are shown in Tables 3 and 4 below.

[0090] Table 3. Third round H chain PCR primers

[0091]

[0092]

[0093] Table 4. Third round kappa chain PCR primers

[0094]

[0095]

[0096] (2) Prepare the third-round PCR reaction system, including: 12.5 μL of 2× Phanta Buffer, 0.5 μL of dNTP (10 mM), 0.5 μL of Phanta polymerase, 1 μL of upstream primer mixture, 1 μL of downstream primer (50 μM), 1.5 μL of the first-round PCR product, and add double-distilled water to make up to 25 μL. The PCR program is set as follows: 95°C pre-denaturation for 3 min, followed by 95°C denaturation for 15 s, 55 / 57°C annealing for 15 s, 72°C extension for 30 s, 35 or 40 cycles, and finally 72°C final extension for 10 min. The PCR products were used in subsequent experiments to construct antibody heavy and light chain expression vectors.

[0097] 5. Construction of antibody heavy and light chain expression vectors

[0098] (1) The cloned PCR product was gel-recovered and homologously recombined with a linearized vector (using Vazyme C112). The linearized vector includes a heavy chain linearized vector and a light chain linearized vector. The heavy chain linearized vector was obtained by double digestion (AgeI, SalI) of the vector AbVec2.0-IGHG1 (Addgene) containing the human IgG1 heavy chain constant region, and the light chain linearized vector was obtained by double digestion (AgeI, BsiWI) of the vector AbVec1.1-IgKC (Addgene) containing the light chain kappa chain constant region.

[0099] (2) Transform XL10 competent cells with the recombinant product and culture overnight.

[0100] (3) On the second day, single colonies were picked from the transformation plates. The plasmids were extracted and, after being identified as correct by enzyme digestion, they were used for transfection to express antibodies, thus obtaining antibody heavy chain expression vectors and antibody light chain expression vectors.

[0101] Example 4: Purification of LN1F9 and LN1D1 Antibodies

[0102] The plasmids of the correct antibody heavy and light chains were co-transfected into Expi293F cells, with a transfection ratio of heavy chain: light chain = 1:1.2. The transfection reagent was PEI, cell:plasmid:PEI = 2 (mL): 3 (μg): 4.5 (μL). Six days after transfection, the cell culture supernatant was collected and centrifuged at 8000×g for 20 minutes. The supernatant was filtered with a 0.45μm filter membrane and incubated with protein A beads (Tiandi Renhe). During purification, the impurities were first washed with PBS, and then eluted with 0.1M Glycine at pH 2.7. The eluate was immediately neutralized with 1M Tris-HCl at pH 9.0. The buffer was changed 2 to 3 times with a 30kDa ultrafiltration tube to PBS buffer, and then it can be used for the next experiment or flashed with liquid nitrogen and stored in a -80°C refrigerator. The SDS-PAGE gel images of the purified LN1F9 and LN1D11 antibodies are shown below. Figure 4 As shown in A and 4B, it shows that LN1F9 and LN1D11 antibodies were successfully purified.

[0103] Example 5 Binding curves of LN1F9 and LN1D1 antibodies with NiV G protein

[0104] 1. Antigen coating: Dilute NiV G head domain protein to 3 μg / mL in coating buffer and add 50 μL / well to a 96-well microtiter plate (Corning). Coat overnight at 4°C.

[0105] 2. Wash the plate: Wash the plate with PBS-T buffer, 250 μL / well, four times;

[0106] 3. Blocking: Block with PBS-T + 1% BSA, 50 μL / well, incubate at 37°C for 2 h;

[0107] 4. Add diluted primary antibody: 50 μL / well, 37°C, 2h (antibody diluted with blocking buffer, starting concentration 5 μg / mL, 5-fold gradient dilution)

[0108] 5. Wash the plate: Wash the plate with PBS-T buffer, 250 μL / well, four times;

[0109] 6. Add secondary antibody: HRP Goat anti-Human IgG (H+L), diluted 1:20,000 with blocking solution, 50 μL / well, and incubate at 37°C for 1 hour.

[0110] 7. Wash the plate: Wash the plate with PBS-T buffer, 250 μL / well, four times;

[0111] 8. Add TMB colorimetric solution (New Cymer), 50 μL / well, incubate in dark at room temperature for 10 min, and stop by adding 1 M HCl;

[0112] 9. Detect OD450 with microplate reader

[0113] GraphPad nonlinear regression and three-parameter fitting were used to draw the standard curve, and the median effect concentration (EC) of the monoclonal antibody was calculated based on the standard curve and the dilution factor. 50 ), the results are shown in Figure 5 . Figure 5 In the figure, the curves show that both LN1F9 and LN1D11 antibodies bind specifically to NiV G and exhibit a dose-response relationship.

[0114] Example 6 Neutralization curves of two monoclonal antibodies against VSV-NiV-M, VSV-NiV-B or VSV-HeV

[0115] 1. VSV-NiV-M and VSV-NiV-B pseudovirus packaging

[0116] The full-length NiV or HeV G (G protein of NiV-M strain: NP_112027.1, G protein of NiV-B strain: AAY43916.1, HeV G protein: NP_047112.2) and F (F protein of NiV-M strain: NP_112026.1, F protein of NiV-B strain: AAY43915.1, and to improve virus titer, we introduced two point mutations, S207L and G252D, into the F protein of NiV-B strain; HeV F protein: NP_047111.2) genes were inserted into the pCAGGS expression vector through the EcoRI and NotI restriction sites, respectively. The plasmids containing the G and F genes were co-transfected into HEK293T cells using Genetwin as the transfection reagent. Six hours after transfection, replace the culture medium with fresh DMEM + 10% FBS + 1% P / S and incubate at 37°C, 5% CO2 for 24 hours. After 24 hours, infect the cells with diluted VSV-ΔG-eGFP tool virus (1:10 dilution in DMEM + 4% FBS). Infect for approximately 4-6 hours, discard the supernatant, wash twice with PBS, and then replenish the culture medium containing VSV-G monoclonal antibody-I1 (DMEM + 4% FBS + 1% P / S). Incubate at 37°C, 5% CO2 for another 24 hours, then collect the viral supernatant by centrifugation. Aliquot the collected viral supernatant and store in a -80°C freezer.

[0117] 2. Two monoclonal antibodies neutralize VSV-NiV-M, VSV-NiV-B or VSV-HeV pseudovirus infection

[0118] (1) Plating, VeroE6 cells were digested and diluted to 1.5×10 5 The cells were plated into a 96-well cell culture plate at a density of 100 μL per well and placed in a 37°C, 5% CO2 incubator for 24 h.

[0119] (2) VSV-NiV-M, NiV-B or HeV pseudoviruses were diluted and mixed with serially diluted antibodies in a 96-well U-bottom plate. The mixture was incubated at 37°C for 1 hour, and then the virus-antibody mixture was added to the VeroE6 cells plated in a 96-well plate the day before. After culturing in a 37°C, 5% CO2 incubator for 24 hours, the cells were fixed with 4% paraformaldehyde, and the green fluorescent spots were counted using CTL-S6 Ultra M2. GraphPad Prism was then used to draw the infection inhibition curve and calculate the half-maximal inhibitory concentration (IC). 50 ).

[0120] The results are as follows Figure 6 IC of LN1F9 monoclonal antibody against VSV-NiV-M pseudovirus 50 =0.0719 ng / mL, neutralizing the IC of VSV-NiV-B pseudovirus 50 =0.108ng / mL, neutralizing IC of VSV-HeV pseudovirus 50 =42.03μg / mL. IC of LN1D11 monoclonal antibody neutralizing VSV-NiV-M pseudovirus 50 =0.657ng / mL, neutralizing the IC of VSV-NiV-B pseudovirus 50 =0.597ng / mL.

[0121] Example 7 Two monoclonal antibodies neutralize NiV-M, NiV-B or HeV live viruses

[0122] Antibodies were serially diluted 3-fold in DMEM containing 2% FBS, and 100 TCID 50 NiV-M live virus, 50TCID 50 NiV-B live virus or 100TCID 50 The live HeV virus was incubated in a 37°C, 5% CO2 incubator for 1 hour. After incubation, the virus-antibody mixture was incubated with pre-plated Vero E6 cells at 37°C, 5% CO2 for 1 hour. Four replicate wells were set for each dilution. On the fifth day after virus infection, the cells in the wells were scored for cytopathic effect (CPE) and the IC 50 .

[0123] The results are as follows Figure 7 As shown, the IC of LN1F9 monoclonal antibody against live NiV-M virus 50 =0.084μg / mL, IC for neutralizing live NiV-B virus 50 =0.81μg / mL, IC for neutralizing live HeV virus 50 =38μg / mL. IC of LN1D11 monoclonal antibody neutralizing NiV-M live virus 50 =0.020μg / mL, IC for neutralizing live NiV-B virus 50 =0.0610μg / mL.

[0124] Example 8 Animal protection experiment of LN1F9 and LN1D11 antibodies

[0125] In the antibody prevention virus infection experiment, 6-8 week old Syrian golden hamsters (6 / group) were intraperitoneally injected with 30 mg / kg of the antibody purified in Example 4. 24 hours later, 1000 LD 50 NiV-M virus, the survival status of hamsters was observed every day. The survival rate of hamsters in LN1F9 and LN1D11 antibody groups was 100%, while all hamsters in the control group died. Figure 8 As shown in A.

[0126] In the antibody treatment of viral infection experiments, 6-8 week-old Syrian golden hamsters (6 / group) were intraperitoneally injected with 1000LD 50 NiV-M virus was injected intraperitoneally 1 day and 3 days later, the purified LN1F9 and LN1D11 antibodies in Example 4 were injected twice at 15 mg / kg, for a total of 30 mg / kg. The survival status of the hamsters was observed every day. The survival rate of the mice in the LN1F9 antibody group was 100%, and the survival rate of the mice in the LN1F9 antibody group was 83.3%, while all the mice in the control group died. Figure 8 As shown in B.

[0127] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0128] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0129] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A protective monoclonal antibody or antigen-binding fragment thereof targeting Nipah virus G protein, characterized in that: The protective monoclonal antibody or antigen-binding fragment thereof targeting Nipah virus G protein comprises a heavy chain variable region and a light chain variable region, and includes one of the following monoclonal antibodies: Monoclonal antibody LN1F9 or an antigen-binding fragment thereof: capable of recognizing the G protein of Nipah virus and Hendra virus, wherein the heavy chain variable region of the antibody comprises complementarity determining regions 1-3 as shown in the amino acid sequences of SEQ ID NO: 1-SEQ ID NO: 3, respectively; The light chain variable region has complementarity determining regions 1-3 with amino acid sequences as shown in SEQ ID NO: 6 to SEQ ID NO: 8, respectively; Monoclonal antibody LN1D11 or an antigen-binding fragment thereof: capable of recognizing the G protein of Nipah virus, wherein the heavy chain variable region of the antibody comprises complementarity determining regions 1-3 of the amino acid sequences set forth in SEQ ID NO: 15 to SEQ ID NO: 17, respectively; The light chain variable region thereof has complementarity determining regions 1-3 with amino acid sequences shown in SEQ ID NO: 20 to SEQ ID NO: 22, respectively.

2. A protective monoclonal antibody or antigen-binding fragment thereof targeting Nipah virus G protein according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody LN1F9 or its antigen-binding fragment is shown in SEQ ID NO: 4; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 9; The amino acid sequence of the heavy chain variable region of the monoclonal antibody LN1D11 or its antigen-binding fragment is shown in SEQ ID NO: 18; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

23.

3. The protective monoclonal antibody or antigen-binding fragment thereof targeting Nipah virus G protein according to claim 1, characterized in that: The monoclonal antibody LN1F9 or an antigen-binding fragment thereof comprises a heavy chain as shown in SEQ ID NO: 5, and a light chain with an amino acid sequence as shown in SEQ ID NO: 10; The monoclonal antibody LN1D11 or an antigen-binding fragment thereof comprises a heavy chain as shown in SEQ ID NO: 19 and a light chain as shown in SEQ ID NO:

24.

4. The protective monoclonal antibody or antigen-binding fragment thereof targeting Nipah virus G protein according to any one of claims 1 to 3, characterized in that: The monoclonal antibody or antigen-binding fragment thereof further comprises: Fab, Fab', Fab'-SH, scFv, F(ab')2 with the same antigen-binding fragment; or a heavy chain variable region having an amino acid sequence that is at least 80% homologous to the heavy chain variable region; and a light chain variable region having an amino acid sequence that is at least 80% homologous to the light chain variable region; Or an antibody obtained by connecting a tag to the N-terminus and / or C-terminus of the monoclonal antibody; Alternatively, the monoclonal antibody is a murine antibody, a humanized antibody, a bispecific / trispecific antibody or a chimeric antibody.

5. A polypeptide comprising the amino acid sequence of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. A nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1 to 4, characterized in that: The nucleic acid molecules include a nucleic acid molecule encoding the heavy chain variable region and a nucleic acid molecule encoding the light chain variable region.

7. The nucleic acid molecule according to claim 6, characterized in that The nucleic acid molecule comprises at least one of the following: A nucleic acid molecule encoding the monoclonal antibody LN1F9 or an antigen-binding fragment thereof: having a heavy chain variable region as shown in SEQ ID NO.11 or a heavy chain as shown in SEQ ID NO.12, and having a light chain variable region as shown in SEQ ID NO.13 or a light chain as shown in SEQ ID NO.14; A nucleic acid molecule encoding the monoclonal antibody LN1D11 or an antigen-binding fragment thereof: having a heavy chain variable region as shown in SEQ ID NO.25 or a heavy chain as shown in SEQ ID NO.26, and having a light chain variable region as shown in SEQ ID NO.27 or a light chain as shown in SEQ ID NO.

28.

8. An expression vector comprising the nucleic acid molecule according to any one of claims 6 to 7, characterized in that: The expression vector is capable of expressing the nucleic acid in a prokaryotic or eukaryotic host cell.

9. An engineered bacterium comprising the expression vector according to claim 8.

10. A eukaryotic host cell comprising the expression vector of claim 8.

11. Use of the protective monoclonal antibody targeting Nipah virus G protein according to any one of claims 1 to 4, or the polypeptide according to claim 5, or the nucleic acid molecule according to any one of claims 6 to 7, or the expression vector according to claim 8, or the engineered bacteria according to claim 9, or the eukaryotic host cell according to claim 10 in the preparation of a drug for preventing and treating Nipah virus or in the preparation of a reagent for detecting Nipah virus.

12. Use of the monoclonal antibody LN1F9 or an antigen-binding fragment thereof in any one of claims 1 to 4 in the preparation of a medicament for preventing and treating Hendra virus infection or in the preparation of a reagent for detecting Hendra virus.

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