CN117402238B

CN117402238B - Broad-spectrum neutralizing antibody targeting henipa virus fusion protein DI and DIII region and application thereof

Info

Publication number: CN117402238B
Application number: CN202311703202.8A
Authority: CN
Inventors: 范鹏飞; 于长明; 任怡; 刘子璇; 李建民; 迟象阳; 房婷; 张冠英; 孙冰洁; 陈郑珊; 陈薇
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2023-12-12
Filing date: 2023-12-12
Publication date: 2024-03-05
Anticipated expiration: 2043-12-12
Also published as: CN117402238A

Abstract

The invention discloses a monoclonal antibody targeting a fusion glycoprotein DI and a DIII region of henipa virus. The antibody is obtained by screening through a flow separation-single cell PCR technology and consists of a monkey variable region and a human constant region, wherein both the light chain and the heavy chain of the monkey variable region have unique CDR regions. The antibody provided by the invention targets the same region of the F trimer protein, shows excellent antigen binding capacity, and has nanomolar or subnanomolar high affinity with the F protein of hendra virus and nipah virus. The antibody can effectively recognize the F protein with a natural structure displayed on the surface of the membrane, and can effectively neutralize pseudoviruses of various henipa viruses; structural analysis showed that the antibodies act on both the DI and DIII domains of protein F. The invention also discloses application of the monoclonal antibody of the anti-henipa virus fusion glycoprotein F in preparing henipa virus therapeutic drugs.

Description

Broad-spectrum neutralizing antibody targeting henipa virus fusion protein DI and DIII region and application thereof

Technical Field

The invention discloses a monoclonal antibody, and belongs to the technical field of polypeptides.

Background

Nipah virus (NiV) and Henla virus (HeV) are negative-strand single-stranded RNA viruses of the genus Henla virus (HNVs) of the family Paramyxoviridae, which cause encephalitis and respiratory symptoms in humans, with a mortality rate between 50% and 100%. Henipa also includes gana virus (GhV), cedar virus (CedV), and in recent years, the chinese ink river virus (MojV) and langv virus (LayV) have emerged in our country. The natural host of henipavirus is fruit bat, and can also infect livestock such as pigs and horses. Humans are infected primarily by contact with body fluids of the infected animal or human.

Currently, niV genome sequence analysis determines two major branches: m genotype and B genotype, wherein M genotype comprises NiV isolate from Malaysia (NiV _MY ) The B genotype includes the bangladesh NiV isolate (NiV _BD ) And Indian NiV isolates (NiV _I ). The three strains have high homology, niV _BD The infectivity and pathogenicity of the branches are obviously higher than that of NiV _MY Branching. The active range of the henipav host covers the southern area of China, and two novel henipav viruses, namely the ink river virus and the Langya virus, are discovered in recent years in China, so that potential threat is generated to the biological safety of China.

The henipav genome is about 18 kb in length and consists of six transcriptional units and untranslated regions at the 3 'and 5' ends, encoding 6 structural proteins in total, respectively, nucleoprotein (nucleocapid protein), phosphoprotein (phosphoprotein), membrane protein (matrix protein), fusion glycoprotein (fusion protein), attachment glycoprotein (attachment protein), and polymerase protein (large protein). Wherein, the P gene additionally codes 3 non-structural proteins of V protein, W protein and C protein by means of mRNA editing, guanine ribonucleotide insertion, variable reading frame and the like. Invasion of cells by hendra virus and nipah virus (HNVs), which are highly deadly to humans, is mediated by both membrane surface attachment glycoprotein (G) and fusion glycoprotein (F). G is a type II membrane protein in tetrameric form and F is a type I membrane protein in trimeric form. After the head domain of the G protein is combined with the liver petunidin B2/B3 receptor on the surface of the host cell, a trigger signal is transmitted to the F protein, and the F protein is converted into a post-fusion conformation from a pre-fusion conformation, so that the virus and the host membrane are fused. Syncytia formation is a typical phenomenon of infection of host cells by nipah virus and hendra virus.

The high risk of henipav determines that the need for live virus related manipulations is limited to the fourth biosafety laboratory 4 (BSL-4), which limits vaccine and antibody development. Currently, there is no approved vaccine or treatment for humans against HNVs. The development strategy of vaccines and antibodies mainly targets G protein and F protein, and only G protein-related VSV vector vaccines and mRNA-1215 vaccines have been entered into clinical phase I trials to date. Related researches evaluate that the treatment effect of some broad-spectrum antiviral small molecule drugs against henipa virus in animals is not ideal, and the broad-spectrum antiviral small molecule drugs can be well protected only by high-dose continuous administration for more than 10 days. In contrast, the antibody medicine has the characteristics of strong specificity and good safety, exerts good protective efficacy in animals, and is an ideal choice for treating henipa virus diseases. M102.4 mab targeting the G protein receptor binding region was able to effectively protect ferrets, african green monkeys, and was also urgently applied to 15 individuals exposed to HeV or NiV infection at high risk. Currently, m102.4 antibodies have completed phase I clinical. Murine antibody 5B3 targeting HNVs F protein also effectively protects animals challenged with lethal doses of virus. The limited number of antibodies, together with the in vitro isolated escape mutants of m102.4 and 5B3, underscores the technical need for developing more and more potent broad-spectrum henipa virus candidate antibodies, and the object of the present invention is to provide a broad-spectrum antibody against henipa virus.

Disclosure of Invention

With the above objects in mind, the present invention provides, first, a monoclonal antibody targeting DI and DIII regions of henipav fusion proteins (also referred to as F proteins or fusion glycoproteins F in the present invention), the heavy chain variable region CDR1, CDR2 and CDR3 amino acid sequences and the light chain variable region CDR1, CDR2 and CDR3 amino acid sequences of the antibodies are shown in any one of the following sequence combinations, respectively:

positions 26-34, 53-59 and 98-118 of SEQ ID NO. 1 and positions 27-32, 50-52 and 89-97 of SEQ ID NO. 3, or

Positions 26-33, 51-58, 97-119 of SEQ ID NO. 5 and positions 27-32, 50-52, 89-97 of SEQ ID NO. 7.

In a preferred embodiment, the amino acid sequence of the antibody heavy chain variable region and the amino acid sequence of the antibody light chain variable region are each as shown in any one of the following sequence combinations:

SEQ ID NO. 1 and SEQ ID NO. 3, the antibody of one specific embodiment having the amino acid sequence of the heavy chain variable region and the amino acid sequence of the antibody light chain variable region in the present invention is designated as "5C8", or

SEQ ID NO. 5 and SEQ ID NO. 7 an antibody of a specific embodiment having the amino acid sequence of the heavy chain variable region and the amino acid sequence of the antibody light chain variable region in the present invention is designated as "4F9".

In a more preferred embodiment, the amino acid sequence of the heavy chain constant region of the antibody is shown in SEQ ID NO. 9 and the amino acid sequence of the light chain constant region of the antibody is shown in SEQ ID NO. 11.

Next, the present invention provides a polynucleotide encoding the heavy chain and the light chain of the above monoclonal antibody, a polynucleotide sequence encoding the heavy chain variable region of the antibody and a polynucleotide sequence encoding the light chain variable region of the antibody, respectively, as shown in any one of the following sequence combinations:

SEQ ID NO. 2 and SEQ ID NO. 4, a monoclonal antibody having the heavy chain variable region and light chain variable region coding sequences of 5C8, or

SEQ ID NO. 6 and SEQ ID NO. 8, the monoclonal antibody having the heavy chain variable region and light chain variable region coding sequences is 4F9.

In a preferred embodiment, the polynucleotide encoding the heavy chain constant region of the antibody has the sequence shown in SEQ ID NO. 10 and the polynucleotide encoding the light chain constant region of the antibody has the sequence shown in SEQ ID NO. 12 (Kappa chain).

Third, the present invention provides a functional element expressing the above polynucleotide encoding the heavy and light chains of a monoclonal antibody.

In a preferred embodiment, the functional element is a linear expression cassette or a mammalian expression vector.

In one embodiment of the invention, a linear expression cassette comprising the full length of the light and heavy chains of an antibody is constructed by ligating the promoter sequences (GenBANK accession number: X03922.1), the coding sequence of the antibody leader peptide, the antibody variable region (amplified from a single cell), the antibody constant region (biosynthesized), and the poly A tail (GenBANK accession number: X03896.1) by overlap extension PCR.

In another embodiment of the invention, the antibody light-heavy chain full length and light chain full length coding genes are cloned using the mammalian expression vector pcDNA3.4.

Fourth, the present invention provides host cells containing the linear expression cassette or mammalian expression vector.

In a preferred embodiment, the cell is a HEK293T cell or an Expi 293F cell.

In one embodiment of the invention, the full length of the light and heavy chains of the antibodies on the linear expression cassette and the full length gene of the light chain are expressed using HEK293T cells as host cells.

In another embodiment of the invention, the full length of the light and heavy chain of the antibody and the full length gene of the light chain on pcDNA3.4 vector are expressed using an Expi 293F cell as a host cell.

Finally, the invention provides application of the monoclonal antibody in preparing a henipa virus disease treatment drug.

The invention adopts a flow separation-single cell PCR technology to obtain a group of strong monoclonal antibodies targeting Huntiepa virus F protein, 5C8 and 4F9 from peripheral blood of rhesus monkeys. The antibodies consist of a monkey variable region and a human constant region, both the light and heavy chains of the monkey variable region having unique CDR regions. The antibody provided by the invention targets the same region of the F trimer protein, shows excellent antigen binding capacity, and has nanomolar or subnanomolar high affinity with the F protein of hendra virus and nipah virus. The antibody can effectively identify the F protein with a natural structure displayed on the surface of the membrane; the antibody can effectively neutralize pseudoviruses of various henipa viruses; the high resolution complex structure of the antigen binding fragment of representative antibody 5C8 with F trimer suggests that the antibody acts on both the DI and DIII domains of F protein. Therefore, the monoclonal antibody of the anti-henipa virus fusion glycoprotein F can be used for preparing henipa virus therapeutic drugs, provides broad-spectrum and specific candidate drugs for broad-spectrum vaccine design, targeted therapy and cocktail therapy development of the antibody, and is beneficial to improving the quick response capability of coping with sudden new infectious diseases.

Drawings

FIG. 1 is a chart of immunogen preparation and purity, particle size and activity detection and analysis;

FIG. 2 shows a serum binding and neutralization titer assay;

FIG. 3 is a flow-sorting NiV sF-specific memory B cell analysis map;

FIG. 4 is a light and heavy chain variable region pairing amplification map of an antibody;

FIG. 5 ELISA screening HNVs sF-specific antibody OD value distribution map;

FIG. 6 shows the distribution of the infection rate of neutralizing antibody cells in the screening of pseudovirus neutralization experiments;

FIG. 7 is a graph of ELISA detection antibody cross-binding activity;

FIG. 8 is a graph showing the detection result of the binding capacity of an antibody to a natural structure F protein displayed on the surface of a membrane;

FIG. 9 is a graph of cross-neutralizing activity of a test antibody for pseudovirus neutralization;

FIG. 10 is a graph of a competition assay for binding of antibodies;

FIG. 11 is a graph of affinity of antibodies to NiV and HeV F proteins;

FIG. 12 NiV _BD -sF/5C8-Fab complex structure schematic.

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are only exemplary and do not limit the scope of the invention in any way, which is defined by the claims.

Example 1 antibody screening

1. Preparation of antigens

Using NiV _BD And full-length sequences of the HeV F protein to prepare mRNA vaccine for primary immunization of animals. Capillary electrophoresis detected transcript, capping product homogeneity with purity greater than 80% (FIGS. 1A, 1B). Liposome nanoparticles of mRNA-HNVs F were then encapsulated, the prepared mRNA-NiV/HeV F-LNP encapsulation efficiencies were 91.5% and 87.4%, respectively, with average particle sizes of 86.1 nm and 88.3 nm, respectively (FIG. 1C). In addition, the recombinant plasmids of the extracellular domain of the F protein of Nipah virus and Hendela virus were constructed, and the soluble F protein (sF) was expressed in an Expi293 system to give the target protein with a purity of 90% or more (FIG. 1D), and the conformation of the purified sF protein was correct by ELISA using the positive antibody 5B3 (FIGS. 1E, 1F).

2. Immune rhesus monkey

Rhesus monkeys were immunized by intramuscular injection, and the primary immunization (day 0) was boosted with 100 μg of each of mRNA-NiV F-LNP and mRNA-HeV F-LNP, and on days 28 and 49 with NiV sF and HeV sF (100 μg sF+0.25 mg aluminum adjuvant+150 μg CpG), respectively. Procoagulant isolated serum was collected on days 0, 21, 28, 35, 42, 49 and 56 followed by detection of antibody binding and neutralization levels, and procoagulant isolated peripheral blood mononuclear cells on day 77 were collected for antibody screening. Day 56 serum has an equal level of high binding (.about.10) to HNVs ⁶ FIG. 2A) and neutralization (≡10) ⁴ Fig. 2B) titer.

3. FITC-labeled F protein

Specific memory B cells need to be sorted by fluorescence-labeled antigen, fluorescein Isothiocyanate (FITC) labeled antigen protein NiV sF method as follows:

(1) FITC (SIGMA, F4274) was dissolved in DMSO at a concentration of 10 mg/mL.

(2) 200. Mu.g of NiV sF protein was replaced with freshly prepared 0.1M sodium carbonate buffer (pH=9.0) using a 0.5 mL ultrafiltration tube with a molecular weight cut-off of 10 kDa.

(3) mu.L of FITC dye was added to the protein solution, mixed well and incubated at 4℃in the dark for 8 h.

(4) Adding NH ₄ Cl was incubated at a final concentration of 50 mM at 4℃in the absence of light for 2 h.

(5) Excess FITC dye in the protein solution is removed by using PBS and a 0.5 mL ultrafiltration tube with a cut-off molecular weight of 10 kDa for a plurality of times, and the marked NiV sF-FITC is stored at 4 ℃ in a dark place after the concentration is measured.

4. Isolation of rhesus peripheral blood mononuclear cells

PBMCs were isolated from the 77 day collected blood samples described in section 2 of this example by Ficoll density gradient centrifugation as follows:

(1) The monkey lymphocyte separation solution is equilibrated to room temperature, and the mixture is shaken and mixed well, and the lymphocyte separation solution with equal volume for collecting blood sample is added to a clean 50 mL centrifuge tube.

(2) The peripheral blood of rhesus monkeys in the anticoagulation tube was diluted and mixed with an equal volume of PBS solution at room temperature.

(3) The peripheral blood (blood sample: PBS: lymphocyte separating liquid ratio is 1:1:1) diluted by 2 times of the separating liquid is slowly paved above the separating liquid, and the boundary between the two liquid surfaces is kept clear in the process.

(4) The prepared sample was centrifuged at 700 g for 25 min, and the acceleration and deceleration were set to 3 gear.

(5) After centrifugation, the plasma homogenate layer was aspirated and the mononuclear cell layer was carefully transferred to a clean centrifuge tube.

(6) PBS was added to a total volume of 40 mL, and the mixture was centrifuged at 600 g for 5 min, and the acceleration/deceleration was set to 9 gear.

(7) The supernatant was discarded and the PBS wash repeated 1 time. Cells were resuspended in 400. Mu.L PBS and counted after filtration through a 40 μm cell sieve.

(8) Diluting cells to 1X 10 ⁷ cells/mL, single-stained tube and naked cell control tube were added with 100. Mu.L each, and the remaining cells were all added to the sorting tube.

5. Flow sorting NiV sF protein specific memory B cell

(1) Fluorescent antibodies were added to each tube as shown in Table 1.

(2) The cells were incubated at 4℃in the dark for 1 h.

(3) 2 mL of FPBS was added to each tube, and the mixture was centrifuged at 800 g and 4℃for 5 min.

(4) The washing was repeated 1 time.

(5) The supernatant was discarded, the single-stained tube and the control tube were resuspended with 400. Mu.L of FPBS, and the sorting tube was added with the appropriate amount of FPBS and placed at 4deg.C in the dark for sorting.

TABLE 1 amount of fluorescent antibody

(6) Individual memory B cells specific for F protein were sorted using a cell sorter (SONY, MA 900). Firstly, the machine is put on a control tube and a single dyeing tube, and the compensation is adjusted. Cells were then sorted, and a loop gate protocol was set: the lymphocyte is circled by SSC and FSC, designated gate 1 (fig. 3A); selected cells in door 1 were de-adherent by FSC-H and FSH-A; perCP and APC circles out CD3 ^– CD19 ⁺ Is designated as gate 2 (FIG. 3B); selected cells in door 2 were circled to CD27 by PE-Cy7 and PE ⁺ IgG ⁺ Is designated as gate 3 (FIG. 3C); selected cells in door 3 were circled by FITC to give NiV sF ⁺ Is set as gate 4 (fig. 3D); cell population in door 4 (CD 3 ^– CD19 ⁺ CD27 ⁺ IgG ⁺ NiV sF ⁺ ) Is a specific memory B cell.

(7) Individual cells were sorted into 96-well PCR plates, and 20 μl of de-rnase water and 20U rnase inhibitor (0.5 μl) were added in advance per well.

(8) The 96-well plate was rapidly cooled in liquid nitrogen and then placed in dry ice for subsequent single cell PCR experiments.

Results: co-sorting to obtain 564 NiV sF specific memory B cells with surface molecular marker of CD3 ^– CD19 ⁺ CD27 ⁺ IgG ⁺ NiV sF ⁺ (FIG. 3).

6. Single cell PCR amplified antibody variable region gene

(1) Single cell reverse transcription PCR

Reverse transcription PCR systems were prepared as shown in Table 2.

TABLE 2 reverse transcription-PCR reaction System

Reaction conditions: 42 ℃ for 10 min;25 ℃ for 10 min;50 ℃ for 60 min;94℃for 5 min.

(2) First round nested PCR

The first round of nested PCR reactions was performed using TransStart Taq DNA polymerase with reverse transcription PCR products as templates, the system is shown in Table 3 and the primer sequences are shown in Table 4.

TABLE 3 first round nested PCR reaction System

Reaction conditions: firstly, 95 ℃ for 5 min, and secondly, 40 cycles of 95 ℃ and 30s; 57 ℃,30s;72 ℃,45 s; finally, at 72℃for 10min.

TABLE 4 first round nested PCR reaction primers

（SEQID NO.13-49）

(3) Second round nested PCR

The first round of nested PCR products were used as templates, and a second round of nested PCR reactions was performed using TransStart Taq DNA polymerase with the system shown in Table 5 and the primer sequences shown in Table 6.

TABLE 5 second round nested PCR reaction System

TABLE 6 second round nested PCR reaction primers

（SEQID NO.50-88）

(4) Capillary electrophoresis

After the nested PCR, the amplified products were subjected to capillary electrophoresis using QIAxcel DNA Fast Analysis Cartridge, and 192 paired antibody light and heavy chain variable region sequences were selected (FIG. 4). The positive clones were sequenced and the antibody variable region sequences obtained by sequencing were analyzed using Vector NTI software and IMGT website.

7. Expression of antibodies by Linear expression cassettes

A linear expression cassette containing the entire length of the light and heavy chain of the antibody was constructed by ligating the promoter sequence (GenBANK accession number: X03922.1), the coding sequence of the antibody leader peptide, the antibody variable region (amplified from a single cell), the antibody constant region (biosynthesized) and the poly A tail (GenBANK accession number: X03896.1) by overlap extension PCR. The specific process is as follows:

(1) Amplifying the promoter-leader fragment

The PCR reaction was performed according to the reaction system and conditions shown in Table 7, and the target fragment was cut and recovered after agarose gel electrophoresis of the PCR product.

Reaction conditions: firstly, 95 ℃ for 5 min; second, 95 ℃ for 30 cycles, 30s;60 ℃,30s;72℃for 1 min, and finally, 72℃for 10min.

TABLE 7 amplified promoter-leader fragment System

(2) Amplification of constant region-PolyA tail fragments

The PCR reaction was performed according to the reaction system and conditions shown in Table 8, and after the completion of the PCR, agarose gel electrophoresis was performed to cut the target fragment and recover the target fragment.

TABLE 8 amplification of constant region-poly A tail fragment System

Reaction conditions: firstly, 95 ℃ for 5 min, and secondly, 30 cycles of 95 ℃ and 30s;60 ℃,30s;72 ℃,2 min, and finally 72 ℃ and 10min.

(3) Amplification of variable region fragments

The PCR reaction was performed according to the system of Table 9 using the single cell second round nested PCR products with successful pairing of the light and heavy chains as templates.

TABLE 9 amplification of variable region fragment systems

Reaction conditions: firstly, 95 ℃ for 5 min, and secondly, 30 cycles of 95 ℃ and 30s;60 ℃,30s;72 ℃,30s, finally, 72 ℃ for 10min.

(4) Amplification linear expression cassette

PCR reactions were performed according to the system of Table 10.

TABLE 10 amplification of linear expression cassette systems

Reaction conditions: firstly, 95 ℃ for 5 min, and secondly, 30 cycles of 95 ℃ for 30s;60 ℃ for 30s;72 ℃,3min, finally, 72 ℃ and 10min.

The amplified product was recovered by an N96 DNA product purification kit and then detected for DNA content using a NanoVue Plus ultramicro spectrophotometer.

8. ELISA screening for antigen-specific antibodies

(1) HEK293T cells were seeded into 96-well plates at 3X 10 per well ⁴ Cells, 200 μl of medium. Cells were cultured until the density reached 70-90% and transfected.

(2) Light and heavy chain linear expression cassettes (0.15. Mu.g each) were diluted with 10. Mu.L of Opti-MEM serum-free medium, added with 0.6. Mu.L of Turbofect transfection reagent, mixed well and incubated for 15 min at room temperature.

(3) The liposome complex was added to a 96-well plate, and the cells were incubated in a incubator at 37℃for 36 h.

(4) ELISA plates were coated the day before ELISA experiments, 100. Mu.L of NiV F and HeV F antigen protein at a concentration of 1. Mu.g/mL were added to each well and incubated overnight at 4 ℃.

(5) After washing the ELISA plates 3 times with a BioTek 405 LS plate washer, 100. Mu.L of blocking solution (PBST with 2% BSA) was added to each well and incubated in an incubator at 37℃for 1 h.

(6) The plate was washed 3 times. mu.L of supernatant was added to each well, and incubated in an incubator at 37℃for 1 h.

(7) The plate was washed 3 times. 100 μl of goat anti-human IgG (HRP) antibody diluted 1:10000 was added and incubated in an incubator at 37℃for 1 h.

(8) The plate was washed 3 times. 100. Mu.L of TMB single-component color development solution was added, and the mixture was developed at room temperature for 6 min.

(9) 50. Mu.L of stop solution was added and the OD values were read on a SpectraMax 190 microplate reader at wavelengths 450-630 nm.

(10) Positive clones were determined with 2.1 times the average value of the negative wells as the cut value.

Results: binding screening of 192 clones to OD _450-630nm When the value is equal to 0.1, the value is the Cutoff value, and 100 strains are identifiedAntigen-specific antibodies (fig. 5). Of these 92 strains had cross-binding activity to NiV sF and HeV sF,4 strains bound only to NiV sF and 4 strains bound only to HeV sF. 56 strains in the cross-reactive antibody have good expression or strong binding capacity to NiV sF and HeV sF, and OD _450-630nm The values are all greater than 1.0.

9. Screening neutralizing antibodies for pseudovirus experiments

(1) As previously described, the linear expression cassette was transfected into 96-well cell culture plates and the cell supernatants were collected after transfection of 36-48 h.

(2) A new 96-well cell culture plate was taken, 90. Mu.L of transfection supernatant and 10. Mu.L of pre-packaged HIV backbone HeV or NiV pseudovirus were added and incubated at 37℃for 1 h.

(3) HEK293T cell count, 100. Mu.L of cell fluid (containing 3X 10) ⁴ Individual cells) was added to the pseudovirus-cell supernatant mixture at a final volume of 200 μl per well. The plates were placed at 37℃with 5% CO ₂ Incubator cultures 48 h.

(4) The medium was carefully aspirated, 50. Mu.L of diluted 1 Xcell lysate was added and lysed by shaking at room temperature for 10min.

(5) mu.L of lysate supernatant was aspirated, transferred to a 96-well white microplate, 50. Mu.L of luciferase substrate (Promega, E1501) was added, and the luminescence value was read in a multifunctional microplate detector.

Results: the 192 clones were subjected to neutralization screening, and 25 rHIV-HNVs cross-neutralizing antibodies (FIG. 6) were initially identified for antibody production and activity evaluation, using the cell infection rate of 60% as a criterion.

Example 2 preparation of antibodies

According to the results of 8 th and 9 th in example 1, 2 HNVs strong cross-binding/neutralizing antibodies (numbered 5C8 and 4F 9) were selected to construct expression plasmids, and mab was prepared. The method comprises the following steps:

1. the heavy chain and light chain linear expression frame full-length gene is usedEcoRI (NEB, R3101) andNoti (NEB, R3189) was double digested and ligated into pcDNA3.4 expression plasmid.

2. The antibody heavy and light chain plasmids were each 15. Mu.g and transfected into 30 mL of the Expi293 System (Life, A14524)，125 rpm，5% CO ₂ 72 and h are cultivated.

3.3000 Xg, centrifugation at 4℃for 15 min to collect the expression supernatant, suction filtration through a 0.22 μm needle filter and affinity purification using rProtein A.

4. The collected antibodies were concentrated with PBS and then assayed for antibody concentration using BCA protein quantification kit (Thermo Scientific, 23225).

EXAMPLE 3 ELISA detection of antibody binding Activity

1. The day before the experiment, a 96-well enzyme-linked plate is coated with 1 mu g/mL of NiV _BD sF and HeV sF, 100 μl per well. The coated ELISA plate was placed in a wet box at 4℃overnight.

2. The experiment was carried out 3 times on the same day with a plate washer, 100 μl of blocking solution was added to each well, and incubation was carried out at 37deg.C for 1 h.

3. The plate washer is washed 3 times. 150 mu L of monoclonal antibody with concentration of 5 mu g/mL is added into the first hole, and 100 mu L of diluent is added into the rest holes. And sucking 50 mu L from the first hole, adding the mixture into the second hole, and similarly, diluting the mixture according to a gradient of 1:3, wherein the final volume of each hole is 100 mu L. Incubate 1h at 37 ℃.

4. The plate washer is washed 3 times. HPR-labeled goat anti-human IgG secondary antibody (Abcam, ab 97225) was diluted 1:10000, 100 μl per well was added to ELISA plates, and incubated at 37deg.C for 1 h.

5. The plate washer is washed 3 times. 100 mu L of TMB single-component color development liquid is added into each hole, and color development is carried out at room temperature in a dark place for 6 min.

6. 50 μl of stop solution was added to each well to stop the reaction, and the ELISA plate was loaded onto the microplate reader to detect OD values at 450-630 nm. Data were imported into GraphPad software and EC was calculated using four-parameter fitting dose-response curves ₅₀ Values.

Results: 5C8 and 4F9 vs NiV _BD sF (fig. 7A) and HeV sF (fig. 7B) have good cross-binding activity. Wherein 5C8 and 4F9 bind EC with NiV sF ₅₀ Values of 0.6 ng and 4.1 ng/mL, respectively; binding to HeV sF EC ₅₀ The values were 1.0 ng/mL and 4.2 ng/mL, respectively. Control antibody 5B3 (Patent No. US15951327B 2) and NiV _BD Binding EC of sF and HeV sF ₅₀ Values of 15.1 ng and 21.9 ng/mL, respectively; 1H8 and 4H3 (PMID: 36932063) bind only NiV sF, EC ₅₀ Values were 28.3 ng and 2.2 ng/mL, respectively; the unrelated control antibody (control mAb) was Ebola virus GP antibody 2G1, not binding NiV _BD sF or HeV sF. Overall, the cross-binding capacity of the 5C8 and 4F9 mabs was superior to the 3-strain F protein antibodies reported in the prior art.

Example 4 flow cytometry detection of antibody binding Capacity to Membrane surface F protein

1. NiV is carried out _BD Full-length F was transfected into HEK293 cells, and after 36 h culture, the cells were digested with PBS containing 0.03% EDTA.

2. The cells were transferred to a 50 mL centrifuge tube, centrifuged at 600 Xg for 5 min, the supernatant carefully decanted, and the appropriate amount of PBS was added to resuspend the cells.

3. Cells were filtered using a 70 μm cell sieve, cell pellet removed, and counted.

4. Diluting cells, 5X 10 per tube of flow tube ⁵ Individual cells, 100 μl volume.

5. 2. Mu.g/mL of antibody was added to each tube, and the volume was 100. Mu.L, and incubated at room temperature for 1 h.

6. 3 mL of PBS was added to each tube, centrifuged at 600 Xg for 5 min, and the supernatant was carefully decanted.

7. PE Mouse Anti-Human IgG (BD 555787) antibodies (5 μl/tube) were diluted with PBS solution and incubated at 1h in 100 μl volumes per tube at room temperature in the dark.

8.3 mL of PBS was added to each tube, centrifuged at 600 Xg for 5 min, and the supernatant was carefully decanted.

9. Cells were resuspended with 200 μl PBS per tube and analyzed on FACSCanto II flow cytometer, 10000 cells per tube were counted.

Results: compared to the negative control antibody 2G1 (0%, ebola virus GP antibody), both 5C8 and 4F9 were able to bind efficiently to the native structural F full-length protein displayed on the cell membrane surface, with PE positive cell populations of 55.9% and 44.0%, respectively (fig. 8).

EXAMPLE 5 test of neutralizing Activity of antibodies in pseudovirus neutralization experiments

NiV using pre-packaged HIV backbone _BD HeV and HeV-g2 pseudoviruses, and the cross-neutralizing activity of the antibodies was evaluated. The evaluation method is as follows:

1. the monoclonal antibody was diluted with DMEM medium, 75. Mu.L of antibody dilution at a concentration of 5. Mu.g/mL was added to the first well of the 96-well cell culture plate, and 50. Mu.L of DMEM medium was added to the remaining wells.

2. mu.L of liquid was aspirated from the first well and added to the second well, mixed well, and so forth, diluted at a 1:3 ratio with a final volume of 50. Mu.L per well.

3. Pseudoviruses were diluted 1:5 in DMEM medium and added to each antibody well at 50 μl per well. Mix well and incubate 1h at 37 ℃.

4. HEK293T cells were counted, 3X 10 ⁵ cells/mL, 100. Mu.L per well.

5. The 96-well cell culture plate was placed in an incubator at 37℃for 48 h.

6. The cell culture plates were removed and the culture medium carefully aspirated. 50 μl of cell lysate was added to each well and lysed by shaking at 350 rpm for 10min.

7. 20 μl of the lysate was pipetted into a 96-well white microplate, 50 μl of luciferase substrate (Promega, E1501) was added, and the luminescence values were read in a multifunctional microplate detector.

8. Calculating the protection rate of the antibody on cells, importing the data into GraphPad software, adopting four parameters to fit a dose-response curve, and calculating the IC ₅₀ Values.

Results: the 5C8 and 4F9 mabs had good cross-neutralizing activity against 3 HNVs pseudoviruses (fig. 9). Wherein, for rHIV-NiV _BD Is of (2) ₅₀ Values were 24.6 ng/mL and 60.2 ng/mL, respectively; IC for rHIV-HeV ₅₀ Values of 37.9 ng and 25.8 ng/mL, respectively; IC for rHIV-HeV-g2 ₅₀ The values were 4.4 ng/mL and 4.2 ng/mL, respectively. Neutralization of 3 pseudoviruses by control antibody 5B3 (Patent No. US15951327B 2) ₅₀ Values 268.6 ng/mL, 85.7 ng/mL and 22.0 ng/mL, respectively; 1H8 and 4H3 (PMID: 36932063) only neutralize rHIV-NiV _BD ，IC ₅₀ The values were 3198.0 ng/mL and 18.2 ng/mL, respectively. Overall, the cross-neutralizing capacity of the 5C8 and 4F9 monoclonal antibodies to 3 HNVs pseudoviruses is superior to that of the 3 strain F protein antibodies reported above, and nearly 100% neutralization can be achieved to 3 HNVs pseudoviruses at a concentration of 1 μg/mL.

Example 6 competitive binding assay of antibodies

The binding competition of the antibodies was detected by ELISA as follows:

1. on the day before the experiment, 200. Mu.g of antibody was labeled using EZ-Link ™ Sulfo-NHS-Biotin at a molar ratio of 1:20 (antibody: biotin) and centrifuged multiple times to remove free Biotin using a 0.5 mL volume ultrafiltration tube with a 30 kDa cut-off. The concentration of the labeled antibody was measured using nanoVue, and the labeled antibody was stored at 4℃in a dark place.

2.96 well ELISA plates were coated with 100. Mu.L of NiV at a concentration of 1. Mu.g/mL per well _BD sF,4℃overnight.

3. On the day of the experiment, after washing the ELISA plates 3 times with a BioTek 405 LS plate washer, 100. Mu.L of blocking solution was added to each well and incubated at 37℃for 1 h.

4. The plate was washed 3 times. mu.L of unlabeled antibody (100 times EC) was added per well ₅₀ Concentration), added laterally in a matrix and incubated at 37℃for 30 min.

5. 50. Mu.L of biotin-labeled antibody (EC) was added to each well ₅₀ Concentration), added longitudinally in a matrix, incubated at 37℃for 30 min in the same order as the blocking antibodies.

6. The plate was washed 3 times. 100. Mu.L of strepitavidin-HRP antibody diluted 1:10000 was added and incubated at 37℃for 1 h.

7. The plate was washed 3 times. 100. Mu.L of TMB single-component color development solution was added, and the mixture was developed at room temperature for 6 min.

8. 50. Mu.L of stop solution was added thereto, and the OD value at a wavelength of 450 to 630nm was read. Competition value= (biotinylated antibody OD value in the presence of competing antibody/biotinylated antibody OD value in the presence of unrelated antibody) ×100%. The competition value is less than 33.3, and the strong competition is judged; > 66.7, judged not to compete; between 33.3 and 66.7 is a weak competition.

Results: there was a strong competition between the 5C8 and 4F9 antibodies and the control antibody 5B3/1H8 (fig. 10), suggesting that their binding epitopes overlap or are spatially close.

Example 7 antibody affinity assay

The affinity of the antibodies to NiV and HeV F proteins was detected by a Biacore T200 instrument using a Protein a chip, the assay was as follows:

1. the antibody was diluted to 1. Mu.g/mL and loaded onto a Protein A chip at a flow rate of 10. Mu.L/min at 120 s.

2. Protein F was serially diluted 2-fold to 0.40 nM starting at a concentration of 100 nM and loaded onto Protein a chips of captured antibodies at a flow rate of 30 μl/min, bound to 120 s, dissociated 900 s.

3. 5 representative curves were selected and the affinity of the antibodies was calculated by the instrument configuration analysis software (K _D ）。

Results: the 5C8 and 4F9 antibodies have high affinity for NiV and HeV F proteins, both on the sub-nanomolar scale (fig. 11). Wherein, the affinities of 5C8 and 4F9 and NiV F are respectively 1.34 pM and 1.26 pM; affinity for HeV F was 27.00 pM and 64.50 pM, respectively.

EXAMPLE 8.5C8 structural resolution of antibody Fab and NiV sF trimer complexes

Selecting a representative antibody 5C8, preparing a Fab fragment, incubating with NiV sF, preparing an antigen-antibody complex by using a gel column, and obtaining NiV with resolution reaching 2.30A by cryoelectron microscope analysis _BD -sF/5C8-Fab complex structure. FIG. 12 is NiV _BD -sF/1D6-Fab complex structural analysis schematic. The results indicate that 5C8-Fab recognizes a conserved epitope located in the F trimer waist DI and DIII interface region, suggesting that this region may serve as a potential target for broad-spectrum vaccines and antibodies.

Claims

1. A monoclonal antibody targeting henipav fusion proteins DI and DIII, characterized in that the antibody heavy chain variable region CDR1, CDR2 and CDR3 amino acid sequences and the light chain variable region CDR1, CDR2 and CDR3 amino acid sequences are shown in any one of the following sequence combinations, respectively:

2. The monoclonal antibody of claim 1, wherein the amino acid sequence of the antibody heavy chain variable region and the amino acid sequence of the antibody light chain variable region are each shown in any one of the following sequence combinations:

SEQ ID NO. 1 and SEQ ID NO. 3, or

SEQ ID NO. 5 and SEQ ID NO. 7.

3. The monoclonal antibody according to claim 2, wherein the amino acid sequence of the heavy chain constant region of the antibody is shown in SEQ ID NO. 9 and the amino acid sequence of the light chain constant region of the antibody is shown in SEQ ID NO. 11.

4. A polynucleotide encoding the heavy and light chains of the monoclonal antibody of any one of claims 1-3, wherein the polynucleotide sequence encoding the heavy chain variable region of the antibody and the polynucleotide sequence encoding the light chain variable region of the antibody are each shown in any one of the following sequence combinations:

SEQ ID NO. 2 and SEQ ID NO. 4, or

SEQ ID NO. 6 and SEQ ID NO. 8.

5. The polynucleotide according to claim 4, wherein the polynucleotide encoding the heavy chain constant region of the antibody has a sequence shown in SEQ ID NO. 10 and the polynucleotide encoding the light chain constant region of the antibody has a sequence shown in SEQ ID NO. 12.

6. A functional element comprising the polynucleotide encoding the heavy and light chains of a monoclonal antibody of claim 5, which functional element is a linear expression cassette or a mammalian expression vector.

7. A host cell comprising the linear expression cassette or mammalian expression vector of claim 6.

8. The host cell of claim 7, wherein the cell is a HEK293T cell or an Expi 293F cell.

9. Use of the monoclonal antibody of any one of claims 1-3 in the manufacture of a medicament for the treatment of henipav virus diseases.