Neutralizing monoclonal antibodies targeting Nipah virus G protein and uses thereof

Abstract

The present invention provides neutralizing monoclonal antibodies targeting Nipah virus 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 three monoclonal antibodies that can specifically bind to NiV G protein. Antibody epitope competition experiments found that the S1E2 and SB10 monoclonal antibodies among these three antibodies recognize new epitopes of NiV G protein that have not been reported before. In vitro neutralization experiments have demonstrated that these three antibodies have high in vitro neutralizing activity, and can neutralize both NiV-M and NiV-B strains, with the characteristics of high expression and good stability, and can be used to prepare virus detection products such as Nipah and Hendra or drugs for preventing and treating Nipah and Hendra virus diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a neutralizing monoclonal antibody targeting Nipah virus G protein and uses 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, but can also lead to fatal respiratory or neurological illnesses, with a case fatality rate of up to 70%. There are two major 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 expanded. In addition to the previously discovered Hendra virus (HeV), new viruses have been discovered in Africa, Australia, Asia, and South America. Mòjiāng virus (MojV) and Langya virus (LayV) have also been newly discovered in my country. 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 mediate receptor binding and host cell entry. 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 systematic research on G protein antigenic epitopes, especially the development of new antibodies that recognize relatively conserved G protein stems and potential epitopes at the head / stem interface. This suggests that there is still a need to develop a group of protective antibodies with broad neutralizing activity, elucidate the mechanism of action of antibodies, and develop and identify new epitopes on NiV surface glycoproteins. This will provide new candidate drugs and strategies for the prevention and control of HNVs infectious diseases 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 three neutralizing monoclonal antibodies targeting Nipah virus G protein and their uses, which can specifically recognize new 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, a neutralizing monoclonal antibody targeting Nipah virus G protein is provided, wherein the monoclonal antibody can recognize Nipah virus G protein, including one or more of monoclonal antibodies S1E2, S2B10, and LN3D3, each of which comprises a heavy chain variable region and a light chain variable region:

[0008] Monoclonal antibody S1E2: Its heavy chain variable region has three complementarity determining regions of the amino acid sequences set forth in SEQ ID NOs: 1 to 3; its light chain variable region has three complementarity determining regions of the amino acid sequences set forth in SEQ ID NOs: 6 to 8; (the sequence of SEQ ID NO: 7 is: STS);

[0009] Monoclonal antibody S2B10: Its heavy chain variable region has three complementarity determining regions of the amino acid sequences shown in SEQ ID NOs: 15 to 17; its light chain variable region has three complementarity determining regions of the amino acid sequences shown in SEQ ID NOs: 20 to 22; (SEQ ID NO: 21 is the sequence: RAS)

[0010] Monoclonal antibody LN3D3: Its heavy chain variable region has three complementarity determining regions (CDRs) with the amino acid sequences shown in SEQ ID NOs: 29 to 31; its light chain variable region has three CDRs with the amino acid sequences shown in SEQ ID NOs: 34 to 36. (The sequence of SEQ ID NO: 35 is YAS.)

[0011] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody S1E2 is shown in SEQ ID NO: 4; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 9;

[0012] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody S2B10 is shown in SEQ ID NO: 18; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 23;

[0013] The amino acid sequence of the heavy chain variable region of the monoclonal antibody LN3D3 is shown in SEQ ID NO: 32; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 37.

[0014] The monoclonal antibody S1E2 comprises a heavy chain as shown in SEQ ID NO: 5 and a light chain as shown in SEQ ID NO: 10.

[0015] The monoclonal antibody S2B10 comprises a heavy chain as shown in SEQ ID NO: 19 and a light chain as shown in SEQ ID NO: 24.

[0016] The monoclonal antibody LN3D3 comprises a heavy chain as shown in SEQ ID NO: 33 and a light chain as shown in SEQ ID NO: 38.

[0017] Furthermore, the monoclonal antibody further comprises:

[0018] Fab, Fab', Fab'-SH, scFv, F(ab')2 with the same antigen-binding fragment;

[0019] An antibody having the same function as the monoclonal antibody 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 at least 80% homologous to the heavy chain variable region; and a light chain variable region having an amino acid sequence at least 80% homologous to the light chain variable region;

[0020] Or an antibody obtained by connecting a tag to the N-terminus and / or C-terminus of the monoclonal antibody;

[0021] Alternatively, the monoclonal antibody may be humanized to obtain an antibody with the same or similar function.

[0022] 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 region 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: 1-6, and then testing the retained function of the encoded altered antibodies using the functional assays described herein.

[0023] The monoclonal antibodies include: mouse antibodies, humanized antibodies, bi / tri-specific antibodies or chimeric antibodies.

[0024] 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.

[0025] In these operations, the encoding V L or V H The 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.

[0026] In the second aspect of the present invention, a polypeptide containing the amino acid sequence is provided.

[0027] 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, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 37, and SEQ ID NO: 38.

[0028] 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.

[0029] 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 light chain variable region (e.g., SEQ ID NO: 13) of the S1E2 antibody, as well as sequences encoding the heavy chain (e.g., SEQ ID NO: 12) and / or light chain (e.g., SEQ ID NO: 14) of the S1E2 antibody. It also includes sequences encoding the heavy chain variable region (e.g., SEQ ID NO: 25) and / or light chain variable region (e.g., SEQ ID NO: 27) of the S2B10 antibody, as well as sequences encoding the heavy chain (e.g., SEQ ID NO: 26) and / or light chain (e.g., SEQ ID NO: 28) of the S2B10 antibody. These include sequences encoding the heavy chain variable region (such as SEQ ID NO: 39) and / or the light chain variable region (such as SEQ ID NO: 41) of the LN3D3 antibody, as well as sequences encoding the heavy chain (such as SEQ ID NO: 40) and / or the light chain (such as SEQ ID NO: 42) of the LN3D3 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 neutralizing 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 neutralizing monoclonal antibodies targeting Nipah virus G protein provided by the present invention, three monoclonal antibodies S1E2, S2B10 and LN3D3 with high neutralizing activity binding to NiV G protein were obtained by screening NiV G-specific binding plasma cells of mice;

[0036] The monoclonal antibodies screened had high in vitro neutralizing activity, among which LN3D3 had an IC of 0.05 for NiV-M pseudovirus with VSV as the backbone. 50The neutralization IC value for rVSV-NiV-B pseudovirus can be as low as 2.5 ng / mL 50 as low as 12.4 ng / mL;

[0037] Epitope competition experiments found that S1E2 and S2B10 antibodies bind to unreported new epitopes on NiV G protein; the three 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

[0038] 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.

[0039] Figure 1 Figures 1 and 2 show Coomassie Brilliant Blue staining and molecular sieve chromatography of NiV G-ferritin nanoparticle immunogen. 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 the NiV G head domain 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 chromatography of NiV G-ferritin nanoparticles; E shows the molecular sieve chromatography of NiV G; and F shows the molecular sieve chromatography of ferritin.

[0040] 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 results.

[0041] 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.

[0042] Figure 4 This is the purification diagram of S1E2, S2B10, and LN3D3 antibodies; Figure 4A is the SDS-PAGE image of S1E2 antibody purification. Figure 4 B is the SDS-PAGE image of S2B10 antibody purification. Figure 4 C is the SDS-PAGE image of LN3D3 antibody purification.

[0043] Figure 5 The binding curves of three monoclonal antibodies to NiV G protein are shown in Figure 2. Figure 5 A is the binding curve of S1E2 antibody and NiV G protein, Figure 5 B is the binding curve of S2B10 antibody and NiV G protein, Figure 5 C is the binding curve of LN3D3 antibody and NiV G protein.

[0044] Figure 6 The neutralization curves of the three monoclonal antibodies against rVSV-NiV-M and rVSV-NiV-B pseudoviruses, respectively; Figure 6 A is the neutralization curve of S1E2 monoclonal antibody against rVSV-NiV-M pseudovirus, Figure 6 B is the neutralization curve of S2B10 monoclonal antibody against rVSV-NiV-M pseudovirus, Figure 6 C is the neutralization curve of LN3D3 monoclonal antibody against rVSV-NiV-M pseudovirus, Figure 6 D is the neutralization curve of S1E2 monoclonal antibody against rVSV-NiV-B pseudovirus, Figure 6 E is the neutralization curve of S2B10 monoclonal antibody against rVSV-NiV-B pseudovirus, Figure 6 F is the neutralization curve of LN3D3 monoclonal antibody against rVSV-NiV-B pseudovirus.

[0045] Figure 7 There are three monoclonal antibodies that neutralize NiV-M and NiV-B live viruses; among them, Figure 7 A is the half maximal inhibitory concentration (IC) of S1E2 monoclonal antibody against live NiV-M and NiV-B viruses, respectively. 50 ), Figure 7 B is the IC of S2B10 monoclonal antibody against live NiV-M and NiV-B viruses, respectively. 50 , Figure 7 C is the IC of LN3D3 monoclonal antibody against live NiV-M and NiV-B viruses, respectively. 50 .

[0046] Figure 8 BLI is used to detect the competition between antibody epitopes. Figure 8 A is a representative curve of BLI epitope competition detection between S1E2 and S2B10, HENV-26, HENV-32, and nAH1.3. Figure 8B is the representative curve of BLI epitope competition detection between S2B10 and S1E2, HENV-26, HENV-32, and nAH1.3. Figure 8 C is a representative curve of BLI epitope competition detection between LN3D3 and HENV-26, HENV-32, and nAH1.3. DETAILED DESCRIPTION

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] Example 1 Construction, expression and purification of NiV G-ferritin nanoparticle immunogen

[0052] 1. NiV G-ferritin Nanoparticle Immunogen Gene Synthesis and Vector Construction

[0053] 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 G protein from the Malaysian strain (GenBanK: NP_112027.1) (hereinafter referred to as NiV G), and the 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 the N8Q mutation, 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 G) and a ferritin expression plasmid (containing a 6×His residue at the N-terminus) were also constructed. These two plasmids are expressed using the same expression vector as the NiV G-ferritin expression plasmid.

[0054] 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 G expression vector) and the ferritin expression plasmid, are shown in SEQ ID NO.43 to SEQ ID NO.45.

[0055] 2. Protein Expression and Purification

[0056] NiV G-ferrtin expression plasmid and NiV G expression vector were transiently transfected into Expi293 cells using polyethyleneimine (PEI, Polyscience). Transfected cells were incubated at 100 rpm, 37°C, and 8% CO2. The cells were replenished every 48 hours after transfection, and the cell supernatant was collected approximately 108 hours after transfection. The supernatant was filtered through a 0.45 μm filter and purified using High Affinity Ni-Charged Resin FF (GenScript). Soluble protein was further purified by size exclusion chromatography (SEC) using a Superose 6 Increase 10 / 300 GL column (Cytiva) or a Superdex 200 Increase 10 / 300 GL column (Cytiva) in 20 mM Tris and 150 mM NaCl, pH 8.0. Purity was verified by SDS-PAGE.

[0057] 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 disrupted approximately 50 times using a Dounce grinder. Cell debris was then removed by centrifugation at 12,000 × g for 40 minutes. The supernatant was filtered through a 0.45 μm filter and purified using High Affinity 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 assessed by SDS-PAGE.

[0058] The purification results are as follows Figure 1 As shown in AC, SDS-PAGE results showed that the purified NiV G-ferritin, NiVG 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 NiVG, indicating that the nanoparticles are successfully assembled.

[0059] Example 2 Mouse immunization process and flow cytometry gate sorting strategy

[0060] 1. Mouse Immunization Process

[0061] Six-week-old BALB / c mice were purchased from Beijing Weitong Lihua. 1 μg or 10 μg of NiV G-Fe 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 triturated using the rubber side of a 2 mL syringe. After trituration, the grinding mesh was rinsed with culture medium, and the suspension was collected. After centrifugation at 500 × g for 4 minutes, the supernatant was removed, and 3 mL of red blood cell lysis buffer was added, followed by incubation 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.

[0062] 2. Flow cytometry sorting gate strategy

[0063] 2.1 Flow staining steps:

[0064] (1) Prepare 8 single-stained tubes of cells (780 APC-Cy7, CD3, 4, 8 AmyCyan-A, CD19 PE-Cy7, IgD PerCP-Cy5-5, CD138 FITC-A, CD95 PE-A, CD19-APC), 0.5×10 6 Cells / tube, 100 μL per tube (50 μL of cells from the 780 single-stained tubes must be specially treated, and 50 μL of cells should be incubated at 65°C for 1 minute before being combined with the original cells at a 1:1 ratio). Add the dye to the 96-well plate containing the single-stained 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.

[0065] (2) The experimental group was the NiV G-Fe 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 minutes, centrifuged, washed twice with 200 μL staining buffer, and then incubated with dye mixture (dye mixture was 780 APC-Cy7, CD3,4,8 AmyCyan-A, CD19 PE-Cy7, IgD PerCP-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.

[0066] 2.2 Flow sorting:

[0067] 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 - CD19 + CD3 / 4 / 8 - IgD - CD95 + CD138 + NiV G + .

[0068] 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 mice in the NiV G-Fe group was significantly increased (0% vs 5.51%).

[0069] Single positive cells were sorted into a 96-well plate containing lysis buffer (lysis buffer formula for one 96-well plate: 7.2 μL Tris + 18 μL RNase (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.

[0070] Example 3 Amplification of Antibody Variable Region Genes from Single B Cells

[0071] 1. Reverse transcription (using Novozymes R211 HiScriptII 1st Strand cDNA Sythesis Kit)

[0072] (1) After sorting, transfer the 96-well PCR plate containing 7 μL of catch buffer in each well from -80°C to ice, let it stand for 5 minutes, and centrifuge it at 400 × g for 30 seconds at 4°C.

[0073] (2) Place the 96-well plate in a matching PCR instrument, heat at 65°C for 5 minutes, and then quickly place it on ice for 2 minutes.

[0074] (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 minutes, followed by reaction at 85°C for 2 minutes.

[0075] 2. First round of PCR

[0076] (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 for each primer. This is the forward primer premix. The first-round PCR primer sequences are shown in Table 1.

[0077] Table 1. First round PCR primers

[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 minutes, followed by 95°C denaturation for 15 seconds, 46°C annealing for 15 seconds, 72°C extension for 1 minute, 50 cycles, and a final extension at 72°C for 10 minutes. 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.

[0083] Table 2. Seq-PCR primers

[0084]

[0085] (2) Prepare the PCR reaction system 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 make up to 12 μL with double-distilled water. The PCR program was set as follows: pre-denaturation at 94°C for 2 minutes, followed by 40 cycles of denaturation at 94°C for 30 seconds, annealing at 57°C for 30 seconds, and extension at 72°C for 20 seconds, and a final extension at 72°C for 10 minutes.

[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) Sequence the monoclonal PCR products with the correct size bands for both IgH and IgK / L using reverse primers. Use the NCBI BLAST or IMGT database to determine if the antibody sequence is available.

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

[0089] (1) Prepare primer premix: Mix equal volumes of mVH01-F to mVH23-F upstream primers (50 μM stock solution, diluted to 1 μM each), mVK01-F to mVK28-F upstream primers (50 μM stock solution, diluted to 1 μM each), and mJH01-R to mJH04-R and mJK01-R to mJK05-R downstream primers (50 μM stock solution, 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] Table 4. Third round kappa chain PCR primers

[0093]

[0094] (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: pre-denaturation at 95°C for 3 minutes, followed by denaturation at 95°C for 15 seconds, annealing at 55 / 57°C for 15 seconds, extension at 72°C for 30 seconds, 35 or 40 cycles, and a final extension at 72°C for 10 minutes. The PCR products were used in subsequent experiments to construct antibody heavy and light chain expression vectors.

[0095] 5. Construction of antibody heavy and light chain expression vectors

[0096] (1) After gel recovery, the cloned PCR product was homologously recombined with a linearized vector (using Vazyme C112). The linearized vectors included 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.

[0097] (2) Use the recombinant product to transform XL10 competent cells and culture overnight.

[0098] (3) On the second day, single colonies were picked from the transformation plates. The plasmids were extracted and identified as correct by enzyme digestion. They were then used for transfection to express antibodies, thus obtaining antibody heavy chain expression vectors and antibody light chain expression vectors.

[0099] Example 4 Purification of S1E2, S2B10, and LN3D3 Antibodies

[0100] The plasmids of the verified 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 = 2:3:4.5. The cell culture supernatant was collected 6 days after transfection and centrifuged at 8000 × g for 20 minutes. The supernatant was filtered with a 0.45 μm filter membrane and incubated with protein A beads (Tian Di Renhe). During purification, the impurities were first washed with PBS, and then eluted with 0.1 M Glycine at pH 2.7. The eluate was immediately neutralized with 1 M Tris-HCl at pH 9.0. The buffer was changed 2 to 3 times with a 30 kDa ultrafiltration tube to PBS buffer, which can be used for the next experiment or stored in a -80°C refrigerator after flashing with liquid nitrogen. The SDS-PAGE gel images of the purified S1E2, S2B10, and LN3D3 antibodies are shown below. Figure 4 As shown in A, B, and C.

[0101] Example 5 Binding curves of three monoclonal antibodies to NiV G protein

[0102] 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.

[0103] 2. Wash the plate: Wash the plate with PBS-T buffer, 250 μL / well, four times;

[0104] 3. Blocking: Block with PBS-T + 1% BSA, 50 μL / well, incubate at 37°C for 2 hours;

[0105] 4. Add diluted primary antibody: 50 μL / well, 37°C, 2 hours (antibody diluted with blocking buffer, starting concentration 5 μg / mL, 5-fold serial dilution)

[0106] 5. Wash the plate: Wash the plate with PBS-T buffer, 250 μL / well, four times;

[0107] 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.

[0108] 7. Wash the plate: Wash the plate with PBS-T buffer, 250 μL / well, four times;

[0109] 8. Add TMB colorimetric solution (New Cymer), 50 μL / well, incubate in the dark at room temperature for 10 minutes, and stop by adding 1 M HCl;

[0110] 9. Detect OD450 with microplate reader

[0111] 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 S1E2, S2B10, and LN3D3 antibodies all bind specifically to NiV G and exhibit a dose-response relationship.

[0112] Example 6 Neutralization curves of three monoclonal antibodies against rVSV-NiV-M and rVSV-NiV-B

[0113] The rVSV-NiV-M and rVSV-NiV-B pseudoviruses were packaged with full-length NiV G (G protein of NiV-M strain GeneBank: NP_112027.1, G protein of NiV-B strain GeneBank: AAY43916.1) and F (F protein of NiV-M strain GeneBank: NP_112026.1, F protein of NiV-B strain GeneBank: AAY43915.1), and in order to improve the virus titer, we introduced two point mutations, S207L and G252D, into the F protein of NiV-B strain. EcoRI and NotI Restriction sites were inserted into the pCAGGS expression vector. Plasmids containing the G and F genes were co-transfected into HEK293T cells using Genetwin transfection reagent. Six hours after transfection, the culture medium was replaced with fresh DMEM + 10% FBS + 1% P / S and incubated at 37°C in a 5% CO2 incubator for 24 hours. After 24 hours, cells were infected with diluted VSV-ΔG-eGFP tool virus (1:10 dilution in DMEM + 4% FBS). After infection for approximately 4-6 hours, the supernatant was discarded, the cells were washed twice with PBS, and then supplemented with culture medium containing the VSV-G monoclonal antibody-I1 (DMEM + 4% FBS + 1% P / S). The cells were incubated at 37°C in a 5% CO2 incubator for another 24 hours, and the viral supernatant was collected by centrifugation. The collected viral supernatant was aliquoted and stored at -80°C.

[0114] 2. Neutralization of rVSV-NiV-M and rVSV-NiV-B Pseudovirus Infections by Three Monoclonal Antibodies

[0115] (1) Plating: VeroE6 cells were digested and diluted to 1.5×10 5The cells were plated at a density of 100 μL / mL in a 96-well cell culture plate, with 100 μL per well, and cultured in a 37°C, 5% CO2 incubator for 24 h.

[0116] (2) NiV-M or NiV-B pseudovirus was 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 Universal M2. GraphPad Prism was then used to draw the inhibition infection curve and calculate the half-maximal inhibitory concentration (IC). 50 ).

[0117] The results are as follows Figure 6 IC of S1E2 monoclonal antibody against VSV-NiV-M pseudovirus 50 =3.3 ng / mL, IC for neutralizing VSV-NiV-B pseudovirus 50 =119.3ng / mL. IC of S2B10 monoclonal antibody neutralizing VSV-NiV-M pseudovirus 50 =8.7 ng / mL, IC for neutralizing VSV-NiV-B pseudovirus 50 =9.2 ng / mL. IC of LN3D3 monoclonal antibody neutralizing VSV-NiV-M pseudovirus 50 =2.5 ng / mL, IC for neutralizing VSV-NiV-B pseudovirus 50 =12.4 ng / mL.

[0118] Example 7 Neutralization of NiV-M and NiV-B Live Viruses by Three Monoclonal Antibodies

[0119] Antibodies were serially diluted 3-fold in DMEM containing 2% FBS, and 100 TCID 50 NiV-M live virus and 50 TCID 50 NiV-B live 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 up for each dilution. On the fifth day after virus infection, the cells in the wells were scored for cytopathic effect (CPE) and the IC was calculated. 50 .

[0120] The results are as follows Figure 7 As shown, the IC of S1E2 monoclonal antibody against live NiV-M virus50 =99.7 μg / mL, IC for neutralizing live NiV-B virus 50 =418.3 μg / mL. IC value of S2B10 monoclonal antibody for neutralizing live NiV-M virus 50 =646.2 μg / mL, IC for neutralizing live NiV-B virus 50 =62.3 μg / mL. IC value of LN3D3 monoclonal antibody for neutralizing live NiV-M virus 50 =0.28 μg / mL, IC for neutralizing live NiV-B virus 50 =0.67 μg / mL.

[0121] Example 8 Epitope Competition between Three Monoclonal Antibodies and Published NiV G Antibodies

[0122] The epitope competition relationship between NiV G mAbs was detected by biolayer interferometry (BLI), and the detection instrument was Octet-Red9 (Pall Forte Bio). First, the antibody was diluted to 10 μg / mL with equilibration buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3mM EDTA, 0.05% Tween-20, 1% BSA) and loaded onto Octect® ProA Biosensors and incubated with the sensor for 10 minutes. After washing in kinetic buffer for 10 seconds, the biosensor tip was immersed in equilibration buffer containing 500 nM NiV-M G head domain protein and incubated for 120 seconds. Then, the biosensor was immersed in kinetic buffer containing competing antibody (10 μg / mL) and incubated for 120 seconds for epitope competition test. The competing antibody is set as the reference control for the antibody itself. If the binding to the competing antibody is higher than the binding to the self-antibody, it is defined as the binding epitope of the competing antibody and the antibody is non-competitive. The representative competition curve is shown as follows. Figure 8 Previous studies have identified several mAbs with strong neutralizing potency against NiV G, including HENV-32, HENV-26, and nAH1.3. These three antibodies bind to different sites on the G protein, so these three antibodies were set as control antibodies in epitope competition experiments.

[0123] The results showed that S1E2 and S2B10 mAbs recognized novel epitopes on NiV G protein that were different from those of the three control antibodies, while the epitopes recognized by LN3D3 mAb overlapped with those recognized by nAH1.3 mAb.

[0124] 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.

[0125] 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.

[0126] 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. Three neutralizing monoclonal antibodies or antigen-binding fragments thereof targeting Nipah virus G protein, characterized in that: The monoclonal antibodies can recognize Nipah virus G protein, including one or more of monoclonal antibodies S1E2, S2B10, and LN3D3, and the monoclonal antibodies or antigen-binding fragments thereof each include a heavy chain variable region and a light chain variable region: Monoclonal antibody S1E2 or an antigen-binding fragment thereof: the heavy chain variable region thereof comprises complementarity determining regions 1-3 as shown in 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 S2B10 or an antigen-binding fragment thereof: the heavy chain variable region thereof comprises complementarity determining regions 1-3 as shown in the amino acid sequences of SEQ ID NO: 15 to SEQ ID NO: 17, respectively; The light chain variable region has complementarity determining regions 1-3 with amino acid sequences as shown in SEQ ID NO: 20 to SEQ ID NO: 22, respectively; Monoclonal antibody LN3D3 or an antigen-binding fragment thereof: the heavy chain variable region thereof comprises complementarity determining regions 1-3 as shown in the amino acid sequences of SEQ ID NO: 29 to SEQ ID NO: 31, respectively; The light chain variable region thereof has complementarity determining regions 1-3 with amino acid sequences shown as SEQ ID NO: 34 to SEQ ID NO: 36, respectively.

2. A neutralizing 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 S1E2 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 S2B10 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; The amino acid sequence of the heavy chain variable region of the monoclonal antibody LN3D3 or its antigen-binding fragment is shown in SEQ ID NO: 32; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

37.

3. The neutralizing monoclonal antibody or antigen-binding fragment thereof targeting Nipah virus G protein according to claim 1, characterized in that The monoclonal antibody S1E2 or an antigen-binding fragment thereof comprises a heavy chain as shown in SEQ ID NO: 5 and a light chain as shown in SEQ ID NO: 10; The monoclonal antibody S2B10 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; The monoclonal antibody LN3D3 or an antigen-binding fragment thereof comprises a heavy chain as shown in SEQ ID NO: 33 and a light chain as shown in SEQ ID NO:

38.

4. A neutralizing 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; The monoclonal antibody has 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 S1E2 antibody 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 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 S2B10 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 a light chain variable region as shown in SEQ ID NO. 27 or a light chain as shown in SEQ ID NO. 28; The nucleic acid molecule encoding LN3D3 has a heavy chain variable region as shown in SEQ ID NO.39 or a heavy chain as shown in SEQ ID NO.40, and a light chain variable region as shown in SEQ ID NO.41 or a light chain as shown in SEQ ID NO.

42.

8. An expression vector comprising the nucleic acid 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 neutralizing 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 infection or in the preparation of a reagent for detecting Nipah virus.

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