CN110028579B

CN110028579B - Monoclonal antibody of anti-nipah virus envelope glycoprotein and application thereof

Info

Publication number: CN110028579B
Application number: CN201910369352.7A
Authority: CN
Inventors: 陈薇; 徐俊杰; 李耀辉; 殷瑛; 宰晓东; 张军; 刘树玲; 李汭桦
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: Institute of Pharmacology and Toxicology of AMMS
Priority date: 2019-05-05
Filing date: 2019-05-05
Publication date: 2020-12-18
Anticipated expiration: 2039-05-05
Also published as: CN110028579A

Abstract

The invention discloses an anti-nipah virus envelope glycoprotein monoclonal antibody 14F8, which has a unique CDR region, has a binding titer of 0.47ng/mL with nipah virus envelope glycoprotein, shows excellent antigen binding activity, can be used for detecting the nipah virus envelope glycoprotein, and can effectively distinguish the nipah virus envelope glycoprotein from Hendra virus envelope glycoprotein. The invention also discloses application of the 14F8 monoclonal antibody in preparing a medicament for treating the Nipah virus disease, wherein the antibody can effectively inhibit the combination of the envelope glycoprotein of the Nipah virus and a cell receptor EFNB2 thereof, and IC₅₀The value is 50ng/mL, the neutralizing activity is enhanced along with the increase of the antibody concentration, and when the antibody concentration exceeds 1 mu g/mL, the inhibition rate of the antibody tends to reach 100 percent, thereby showing the prospect of the 14F8 monoclonal antibody as a candidate therapeutic antibody for the Nipah virus disease.

Description

Monoclonal antibody of anti-nipah virus envelope glycoprotein and application thereof

Technical Field

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

Background

Nipah virus (NiV), a newly emerging highly pathogenic infectious disease pathogen in south asia in recent years, has been classified with Cedar virus (CedPV), Hendra virus (HeV) and Hendra virus (Hendra virus) as well as within the family Paramyxoviridae (family Paramyxoviridae) of the genus hennipah virus. 2015 + 2018, the world health organization continuously lists the virus and pathogens such as Ebola virus and Marburg virus as the most probable virulent pathogens causing serious epidemic situations and being difficult to deal with at present for four years. The Nipah virus disease has strong infectivity, high mortality rate and wide natural host distribution, seriously influences the global public health and threatens the life health of human beings.

In 1998, a new infectious disease, manifested as encephalitis and respiratory symptoms, occurred in swine farmers in malaysia and singapore. A novel virus is first isolated in the cerebrospinal fluid of a patient, and can generate cross reaction with Hendra virus antibodies, the genome of the virus is analyzed to confirm that the virus is a novel Hendra virus, and the virus is named as Nipah virus because the virus is isolated from the Nipah town (town of Nipah) of the state of Perak of the thunderbolt, Malaysia. In outbreaks of nipah virus disease in malaysia, mild respiratory inflammation and encephalitis first develop in pigs, and humans become infected with nipah virus after exposure to the infectious secretions of the affected pigs, and subsequently develop disease. 276 confirmed cases and 105 dead cases in Malaysia and Singapore epidemic outbreaks, and the fatality rate is 38%.

After the first outbreak, malaysia and singapore did not have a new epidemic. Since 2001, there have been many outbreaks of epidemics in india and bangladesh, which are more dispersed and less frequent than in malaysia, resulting in an increased mortality rate (75% on average), and in both bangladesh and indian outbreaks, patients have clinically appeared similar to malaysia but more severe respiratory disease. A total of over 300 diagnosed cases, from 2001 to 2017, with a recent outbreak in 2015, resulting in 9 deaths. Because the epidemic situation is poor, and a complete public health system is not available, the actual number of cases is possibly more. The existing research shows that the main reason of outbreak of epidemic situation in Bangladesh is that the patient eats date palm juice polluted by bat, and also there are also many cases of people transmission, and the transmission mechanism between people is not clear, but virus infection is probably mainly caused by contacting with the body fluid and secretion of the patient. In 2018, in south India, Kalappa outbreaks of Nipah virus epidemic situation, which leads to death of at least 17 people, and prevention and control of Nipah virus arouse wide attention and attention again.

Nipah virus is an enveloped, single-stranded negative-strand RNA virus, the envelope glycoprotein GP playing an important role in its infection. When nipah virus infects cells, GP first binds to the ephrin receptor on the cell membrane, and then triggers a conformational change of Fusion Protein (FP) to form spikes, mediating Fusion of the viral envelope with the cell membrane, resulting in the delivery of the viral nucleocapsid into the cytoplasm. Therefore, blocking the binding of GP to cellular receptors is the main protection mechanism for nipah virus vaccines and antibody drugs. GP binds to host cell membrane proteins, Ephrin-B2 and Ephrin-B3, Ephrin-B2 and Ephrin-B3 molecules belong to the family of surface glycoprotein ligands that bind to Eph and have a high degree of sequence conservation in known sensitive hosts with 95% to 98% amino acid homology. Ephrin-B2 is expressed in arteries, arterioles, and capillaries in a variety of organs and tissues. After the virus attaches to the host cell containing the Ephrin receptor, the F protein undergoes a conformational change, driving a membrane fusion process between the virion and the host cell, resulting in delivery of the viral nucleocapsid into the cytoplasm.

Currently, a variety of candidate vaccines for nipah virus are under development, including recombinant subunit vaccines prepared using hendra virus GP, recombinant viral vector vaccines carrying NIV-GP genes, and the like. The M102.4 monoclonal antibody obtained by screening through a phage surface display library technology shows good neutralizing activity on Nipah virus, and is expected to become a candidate therapeutic antibody. At present, no vaccine or antibody medicament enters a clinical test stage.

The Nipah virus is a new emerging and highly dangerous virulent pathogen, has the risks of being introduced into China and causing serious epidemic situations, and is necessary to screen and prepare the monoclonal antibody of the anti-Nipah virus GP, thereby providing a basis for preparing a Nipah virus detection reagent and a candidate therapeutic antibody. Therefore, the invention aims to provide the anti-nipavirus GP monoclonal antibody with excellent antigen binding activity, and can effectively inhibit the binding of the nipavirus GP with a receptor thereof, thereby providing the application of the anti-nipavirus GP monoclonal antibody in the preparation of drugs for treating the nipavirus diseases.

Disclosure of Invention

Based on the above purpose, the present invention firstly provides a monoclonal antibody against nipah virus envelope glycoprotein, wherein the amino acid sequences of CDR1, CDR2 and CDR3 in the variable region of the antibody light chain are respectively shown as the amino acid sequences at positions 27-37, 55-57 and 94-102 of SEQ ID NO.1, and the amino acid sequences of CDR1, CDR2 and CDR3 in the variable region of the antibody heavy chain are respectively shown as the amino acid sequences at positions 26-33, 51-57 and 96-105 of SEQ ID NO. 5.

In a preferred embodiment, the amino acid sequence of the antibody light chain variable region is shown in SEQ ID NO.1, and the amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO. 5.

More preferably, the amino acid sequence of the antibody light chain constant region is shown as SEQ ID NO.3, and the amino acid sequence of the antibody heavy chain constant region is shown as SEQ ID NO. 7.

Secondly, the invention also provides a gene coding sequence for coding the light chain and the heavy chain of the monoclonal antibody, wherein the gene coding sequence of the light chain variable region of the antibody is shown by SEQ ID NO.2, and the gene coding sequence of the heavy chain variable region of the antibody is shown by SEQ ID NO. 6.

In a preferred embodiment, the gene coding sequence for the light chain constant region of the antibody is represented by SEQ ID No.4 and the gene coding sequence for the heavy chain constant region of the antibody is represented by SEQ ID No. 8.

The present invention also provides an expression vector capable of expressing the nucleotide coding sequence encoding the heavy chain and/or the light chain of the monoclonal antibody.

In a preferred embodiment, the expression vector is pcDNA3.4.

The invention also provides a host cell containing the expression vector.

In a preferred embodiment, the cell is an Expi293 cell.

Finally, the invention provides the application of the monoclonal antibody in preparing a medicament for treating the Nipah virus disease.

Hair brushThe antibody provided by the invention has a unique CDR region, the binding titer of the antibody and the nipah virus envelope glycoprotein is 0.47ng/mL, and the OD value of ELISA is more than 2.0 when the antibody concentration is more than 9.14ng/mL, thereby showing excellent antigen binding activity. The binding titer of the antibody to the envelope glycoprotein of the Hendra virus is 129.66ng/mL, the OD value is only 0.29 at the antibody concentration of 20000ng/mL, and the 14F8 can be used for detecting the envelope glycoprotein of the Nipah virus and can effectively distinguish the envelope glycoprotein of the Nipah virus from the envelope glycoprotein of the Hendra virus. The antibody can effectively inhibit the binding of the Nipah virus envelope glycoprotein and a cell receptor EFNB2, IC₅₀The value is 50ng/mL, the neutralizing activity is enhanced along with the increase of the antibody concentration, and when the antibody concentration exceeds 1 mu g/mL, the inhibition rate of the antibody tends to reach 100 percent, thereby showing the prospect of the 14F8 monoclonal antibody as a candidate therapeutic antibody for the Nipah virus disease.

Drawings

FIG. 1.14F8 chimeric antibody expression vector construction scheme;

FIG. 2 ELISA for the binding of 14F8 to the nipah virus envelope glycoprotein;

FIG. 3 ELISA for detection of 14F8 binding to Hendra virus envelope glycoprotein;

FIG. 4 liquid phase chip assay for the evaluation of neutralizing activity of 14F8 monoclonal antibody.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of the present invention.

Example 1 antibody preparation

1. Immunizing animals

1) The purified NIV-GP recombinant proteins were mixed with equal amounts of Freund's complete adjuvant and injected subcutaneously in the groin of mice at multiple points, each mouse injected with a protein dose of 20. mu.g, for a total of 3 immunizations.

2) The PSCA recombinant protein and Freund's incomplete adjuvant mixture were injected 2 weeks and 4 weeks later, and the method and the amount were the same as those of the 1 st injection.

3) After 2 weeks of the 3 rd immunization, the tail vein blood of the mice was taken to measure the titer of the immunization, which was 1: 1600 or more for the preparation of the fusion, and 3d of the pre-fusion booster immunization (20. mu.g/mouse).

2. Cell fusion and cloning

1) Spleen cells of mice were aseptically harvested, spleen cell suspensions were prepared, fused with Sp2/0 myeloma cells according to a conventional method, and cultured in HAT selective medium.

2) Changing the culture solution to 200 mu l of fresh HAT-containing culture solution on the 4 th day after fusion, detecting cell culture supernatant by ELISA when cell clones grow to the culture hole area of 1/3-1/2, screening positive clones, changing HT culture solution during primary cloning, and subcloning by a limiting dilution method, wherein continuous subcloning is carried out for more than 3 times, and the positive rate reaches 100%.

3. Preparation of ascites

1) Ascites is prepared by a conventional method.

2) The antibody titer of the ascites and the hybridoma cell culture supernatant was measured by ELISA.

4. Sequencing of hybridoma cells

1) Extracting RNA by a Trizol method;

2) carrying out reverse transcription on the RNA to obtain cDNA;

3) amplifying and obtaining heavy chain and light chain variable regions;

4) cloning to pMD18-T vector, sequencing;

5) the sequencing results were aligned using the IMGT/V-QUEST database and further analyzed.

5.14 sequence description of variable region of monoclonal antibody F8

The amino acid sequence of the light chain variable region is shown in SEQ ID NO.1, the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively shown in amino acid sequences at positions 27-37, 55-57 and 94-102 of SEQ ID NO.1, and the gene coding sequence of the light chain variable region is represented by SEQ ID NO: 2, the amino acid sequence of the light chain constant region is shown as SEQ ID NO.3, and the gene coding sequence of the light chain constant region consists of SEQ ID NO: 4, respectively.

The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.5, the amino acid sequences of the CDR1, the CDR2 and the CDR3 of the heavy chain variable region are respectively shown as the amino acid sequences at positions 26-33, 51-57 and 96-105 of the SEQ ID NO.5, the gene coding sequence of the heavy chain variable region is shown as SEQ ID NO.6, the amino acid sequence of the heavy chain constant region is shown as SEQ ID NO.7, and the gene coding sequence of the heavy chain constant region is shown as SEQ ID NO. 8.

6.14 recombinant expression of chimeric antibody of F8 monoclonal antibody

1) The amino acid sequences of the light and heavy chain variable regions of the 14F8 monoclonal antibody and the amino acid sequence of the human IgG1 constant region are combined in sequence, and the chimeric antibody gene is synthesized after codon optimization.

2) tPA signal peptide is added to the 5' end of the synthesized chimeric antibody gene to construct pcDNA3.4 eukaryotic expression plasmid (FIG. 1 is a schematic diagram of the construction of 14F8 chimeric antibody expression vector).

3) 15. mu.g of each of pcDNA3.4-14F8H and pcDNA3.4-14F8L was transfected into 30mL of Expi293 suspension cells at 125rpm with 5% CO₂And culturing for 72 h.

4)3000 Xg, centrifugating for 10min, collecting the expression supernatant, suction-filtering with 0.22 μm needle filter, and purifying by affinity chromatography using rProtein A chromatographic column.

5) The collected antibodies were subjected to a solution change with PBS, and then the antibody concentration was determined with BCA protein quantification kit.

Example 2 ELISA assay for detecting binding Activity of 14F8 monoclonal antibody and Nipah Virus G protein

1) One day prior to the experiment 96 well ELISA plates were coated with 1. mu.g/mL NIV-GP, 200. mu.L per well. The coated enzyme-linked plate was placed in a wet box at 4 ℃ overnight.

2) The experiment was washed 5 times with a plate washer on the day.

3) Add 100. mu.L of blocking solution to each well and let stand at room temperature for 1 hour.

4) Washing the plate for 3 times.

5) 150 μ L of 14F8 mAb at 20 μ g/mL was added to the first well and 100 μ L of the dilution was added to the remaining wells. Aspirate 50 μ L from the first well and add to the second well and so on, dilute each well in a 1:3 gradient to a final volume of 100 μ L. The mixture was allowed to stand at room temperature for 1 hour.

6) Washing with plate washing machine for 5 times.

7) The HPR-labeled goat anti-mouse IgG secondary antibody was diluted at 1:10000 in a diluent, 100. mu.L of each well was added to the corresponding well of the ELISA plate, and the plate was incubated at room temperature for 1 hour.

8) Washing with plate washing machine for 5 times.

9) Adding 100 mu L of TMB single-component developing solution into each hole, developing for 6min, keeping the room temperature away from light, and then adding 50 mu L of stop solution into each hole to terminate the reaction.

10) Detecting OD value at the position of 450-630nm by using a microplate reader, and storing and recording original data.

11) The resulting data were fitted with a four parameter non-linear fit using GraphPad prism7.0 software and antibody binding titers were calculated as cutoff values at 2.1-fold of negative wells. Finally, the binding titer of the 14F8 antibody is calculated to be 0.47ng/mL, and the result is shown in FIG. 2, and the 14F8 monoclonal antibody has good activity of binding to nipah virus GP.

Example 3 ELISA assay to determine the binding Activity of 14F8 monoclonal antibody to the envelope glycoprotein of Hendra Virus

1) A day before the experiment, 96-well enzyme-linked plates are coated with Hendra virus G protein HEV-GP at 1 mu G/mL and 200 mu L of the Hendra virus G protein HEV-GP is coated in each well. The coated enzyme-linked plate was placed in a wet box at 4 ℃ overnight.

2) The experiment was washed 5 times with a plate washer on the day.

4) Washing the plate for 3 times.

6) Washing with plate washing machine for 5 times.

8) Washing with plate washing machine for 5 times.

11) The resulting data were fitted with a four parameter non-linear fit using GraphPad prism7.0 software and antibody binding titers were calculated as cutoff values at 2.1-fold of negative wells.

The results are shown in fig. 3, the binding titer of the antibody to the envelope glycoprotein of hendra virus is 129.66ng/mL, the OD value is only 0.29 at an antibody concentration of 20000ng/mL, and it is shown that 14F8 can be used to distinguish the envelope glycoproteins of nipah virus and hendra virus.

EXAMPLE 4 evaluation of neutralizing Activity of 14F8 monoclonal antibody by liquid-phase chip assay

Ephrinb2 and Ephrinb3 are cell receptors of GP, the key point of blocking the binding of GP and the cell receptors of virus infection is to inhibit the binding of GP and the receptors, and according to various reports at home and abroad, protective serum or neutralizing antibodies can inhibit the binding of GP and the receptors, and the inhibition effect and the protection are related. The experiment of real virus of Nipah and Hendra virus must be carried out in a 4-level biosafety laboratory, and the protective effect of the vaccine or antibody can be evaluated in vitro and under the common biosafety condition through a receptor competitive inhibition experiment.

1) To a black 96-well plate, 14F8 mab was added at different dilution folds.

2) Magnetic microspheres coated with nipavirus GP were added to a 96-well plate in an amount of about 1000 microspheres per well.

3) Ephrinb2 receptor was diluted to 25ng/mL and 10. mu.L was added per well.

4) SAPE was diluted to 12. mu.g/mL, 10. mu.L per well; incubate 30 minutes with shaking at 800 rpm.

5) The 96-well plate was placed on a magnetic stand for 30 seconds, the well liquid was aspirated off, and 100. mu.L of PBS buffer (containing 1% BSA) was added.

6) After 30 seconds of standing, the liquid in the wells was aspirated. The above steps were repeated for two washes.

7) Add 100 u L PBS buffer (containing 1% BSA) heavy suspension microspheres, using a Luminex xmap instrument for detection.

8) Four-parameter non-linear fits were made to the data using GraphPad prism7.0 software and IC50 values were calculated to be 50 ng/mL. The results are shown in fig. 4, the 14F8 monoclonal antibody can effectively inhibit the binding of NIV-GP with its cell receptor EFNB2, the neutralizing activity is enhanced with the increase of the antibody concentration, when the antibody concentration exceeds 1 μ g/ml (the abscissa representing the concentration is log10 value), the inhibition rate tends to reach 100%, and the prospect of the 14F8 monoclonal antibody as a candidate therapeutic antibody for the nipah virus disease is shown. The 14F8 monoclonal antibody can be used as a candidate therapeutic antibody for the Nipah virus disease.

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Claims

1. A monoclonal antibody against nipah virus envelope glycoprotein, characterized in that the amino acid sequences of CDR1, CDR2 and CDR3 in the variable region of the light chain of the antibody are respectively shown as the amino acid sequences at positions 27-37, 55-57 and 94-102 of SEQ ID NO.1, and the amino acid sequences of CDR1, CDR2 and CDR3 in the variable region of the heavy chain of the antibody are respectively shown as the amino acid sequences at positions 26-33, 51-57 and 96-105 of SEQ ID NO. 5.

2. The monoclonal antibody according to claim 1, wherein the amino acid sequence of the antibody light chain variable region is represented by SEQ ID No.1, and the amino acid sequence of the antibody heavy chain variable region is represented by SEQ ID No. 5.

3. The monoclonal antibody of claim 2, wherein the amino acid sequence of the antibody light chain constant region is set forth in SEQ ID No.3 and the amino acid sequence of the antibody heavy chain constant region is set forth in SEQ ID No. 7.

4. A polynucleotide encoding the light and heavy chains of the monoclonal antibody of any one of claims 1-3, wherein the polynucleotide encoding the light chain variable region of the antibody has the sequence shown in SEQ ID No.2 and the polynucleotide encoding the heavy chain variable region of the antibody has the sequence shown in SEQ ID No. 6.

5. The polynucleotide of claim 4, wherein the polynucleotide encoding the light chain constant region of the antibody has the sequence shown in SEQ ID No.4 and the polynucleotide encoding the heavy chain constant region of the antibody has the sequence shown in SEQ ID No. 8.

6. An expression vector for expressing the heavy and light chains of a monoclonal antibody encoded by the polynucleotide of claim 5.

7. The expression vector of claim 6, wherein the expression vector is pcDNA3.4.

8. A host cell comprising the expression vector of claim 7.

9. The host cell of claim 8, wherein the cell is an Expi293 cell.

10. Use of the monoclonal antibody of any one of claims 1-3 for the manufacture of a medicament for the treatment of a nipah virus disease.