CN116621975A - Human monoclonal antibody against Nipah virus G protein and its application - Google Patents

Abstract

The antibody provided by the application can specifically and high-affinity bind with the nipah virus envelope protein, has effective neutralization activity on the nipah virus taking VSV as a framework, can effectively neutralize the nipah virus on a cellular level, and can completely protect hamsters infected with the nipah virus. Which are directed against epitopes different from the neutralizing epitopes reported. Therefore, the series of antibodies are hopefully used as novel specific antibody medicines for preventing and treating the Nipah virus, and provide more choices for preventing and controlling the Nipah virus.

Description

Human monoclonal antibody against Nipah virus G protein and its application

technical field

The invention relates to the technical field of biomedical engineering, in particular to a human monoclonal antibody against Nipah virus G protein and its application.

Background technique

Nipah virus (NiV) is a negative-strand RNA virus belonging to the family Paramyxoviridae and the genus Henipavirus. Nipah virus has a wide host range and can infect multiple species of mammals. In the natural ecosystem, the virus can infect humans through the natural host fruit bats in two ways, one is through the intermediate host poultry to humans, the other is The way is that direct contact between humans and bats leads to infection. Once infected, it can cause acute and highly fatal infectious diseases with the central nervous system and respiratory system as the main lesions. Since the first Nipah virus infection occurred in Malaysia in 1988, the number of Nipah virus infections announced by the World Health Organization has reached 336, and 260 people died, with a fatality rate as high as 77%. Due to its high virulence, wide host range and lack of specific and effective preventive and therapeutic drugs, Nipah virus is classified as a Biosafety Level 4 pathogen.

The Nipah virus genome is an approximately 18 kb single-stranded negative-sense RNA molecule encoding six proteins. Two of the proteins Adhesin G and Fusion protein F are the major surface glycoproteins of pathogens. The G protein is a type II transmembrane glycoprotein that exists as a tetramer on the viral surface. The G-H loop of the cell membrane surface protein erphrinB2/3 is inserted into the hydrophobic groove of the G protein, and through hydrophobic interactions, the virus is attached to the cell surface and mediates the combination of the virus and the cell; the F protein promotes the combination of the virus and the cell in the initial stage of the infection cycle. Fusion of cell membranes, and fusion of membranes of infected cells with membranes of adjacent adjacent cells to form the characteristic syncytia. Adhesin G binds to cell surface receptors and interacts with F, inducing a change in the conformation of the F protein, prompting membrane fusion to occur, thereby releasing the viral ribonucleoprotein complex into the cytoplasm of the host cell.

Phage display technology is to fuse the displayed gene with the phage's own protein gene (gIII or gVIII) and display it on the surface of the phage. It can construct a large-capacity (10 ¹⁰ -10 ¹¹ ) display library for screening, and it has been paid more and more attention by people because of its unique advantages such as fast speed and short cycle. Recombination at the gene level of antibody molecules can obtain a variety of specific murine, humanized and fully human antibody molecules and fragments. The technical field produced by the combination of phage display technology and antibody genetic engineering is currently in a stage of rapid development. This technology enables the development and research of fully human monoclonal antibodies from the basic research stage to the substantive application and development stage. Infectious diseases offer new hope.

Contents of the invention

In view of the problems in the prior art, this application provides a human monoclonal antibody against Nipah virus G protein and its application.

The present invention utilizes human Fab phage display library, using Nipah virus G protein as antigen, panning anti-Nipah virus human antibody, using prokaryotic expression system to express and purify candidate cloned protein, its biological specificity, cell The neutralization activity of pseudovirus and the protective effect on infected animals were identified and evaluated horizontally, and four fully human Nipah virus antibodies NiV41, 41-4, 41-6 and 41-9 were obtained.

Specifically, the heavy chain variable region of the NiV41 antibody contains the amino acid series shown in Seq ID No: 4, and its 26-33, 51-58, and 97-120 respectively contain amino acids such as SEQ ID No: 1-3 The amino acid sequence is shown, while 41-4, 41-6 and 41-9 have the same heavy chain, but different light chains, and the light chain variable region has three complementarity determining regions (CDRs), LCDR1, LCDR2 and LCDR3.

The light chain of NiV41 includes the amino acid sequence shown in SEQ ID No.8, its LCDR1 includes the amino acid sequence shown in SEQ ID No.5, LCDR2 includes the amino acid sequence shown in SEQ ID No.6, and LCDR3 includes the amino acid sequence shown in SEQ ID No.6. Amino acid sequence shown in No.7.

The light chain of 41-4 includes the amino acid sequence shown in SEQ ID No.12, its LCDR1 includes the amino acid sequence shown in SEQ ID No.9, LCDR2 includes the amino acid sequence shown in SEQ ID No.10, and LCDR3 includes the amino acid sequence shown in SEQ ID No.10, LCDR3 includes The amino acid sequence shown in SEQ ID No.11.

The light chain of 41-6 contains the amino acid sequence shown in SEQ ID No.16, its LCDR1 contains the amino acid sequence shown in SEQ ID No.13, LCDR2 contains the amino acid sequence shown in SEQ ID No.14, and LCDR3 contains the amino acid sequence shown in SEQ ID No.14 The amino acid sequence shown in SEQ ID No.15.

The light chain of 41-9 contains the amino acid sequence shown in SEQ ID No.20, its LCDR1 contains the amino acid sequence shown in SEQ ID No.17, LCDR2 contains the amino acid sequence shown in SEQ ID No.18, and LCDR3 contains the amino acid sequence shown in SEQ ID No.18 Amino acid sequence shown in SEQ ID No.19.

The present invention also discloses an antigen-binding fragment, comprising the heavy chain variable region of the aforementioned antibody against the G protein of Nipah virus, and a bispecific antibody, comprising the variable region of the heavy chain of the aforementioned antibody against the G protein of the Nipah virus. variable region, or the aforementioned Nipah virus antibody.

The present invention also discloses that the heavy chain variable region of the antibody against Nipah virus G protein, the Nipah virus antibody and the antigen-binding fragment are useful in the preparation of preventive and therapeutic drugs, detection probes, fusion polypeptides, fusion proteins, Application of immunoconjugates, genetically engineered host cells.

The above-mentioned fully human monoclonal antibody against Nipah virus provided by the present invention is obtained by the following method: first construct a human The Fab phage display library was then used as the antigen in the ectodomain of the Nipah virus envelope protein expressed in the mammalian cell expression system. After four rounds of screening, an enriched and high-affinity clone NiV41 was obtained. The clone was expressed and purified, identified and evaluated, and identified and evaluated for its biological specificity, pseudovirus neutralization activity at the cellular level, and protective effect on infected animals, indicating that the antibody can effectively neutralize Nipah virus. At the same time, an affinity maturation library was constructed by light chain replacement, and the extracellular domain of the Nipah virus envelope protein was used as an antigen again to obtain three enriched and high-affinity clones 41-4 and 41-6 and 41-9. Based on the prediction of the structure of the antigen-antibody complex based on Alpha Fold2 (see Figure 6), this series of antibodies mainly function through the heavy chain, and the heavy chain CDR3 acts on the central cavity of the antigen protein, competing for the binding of the envelope protein G protein and the receptor Binding sites, thereby inhibiting virus infection.

The beneficial effects of the present invention are: the provided antibody can bind Nipah virus envelope protein specifically and with high affinity, has effective neutralizing activity on Nipah pseudovirus with VSV as the backbone, and can effectively neutralize Nipah virus at the cellular level. and Nipah virus, and can completely protect hamsters infected with Nipah virus. The epitope it targets is different from the reported neutralizing epitope. Therefore, this series of antibodies is expected to become a new specific antibody drug for the prevention and treatment of Nipah virus, providing more options for the prevention and control of Nipah virus.

Description of drawings

Figure 1: Expression and purification of Nipah virus envelope protein G protein. The target protein was detected by polyacrylamide gel electrophoresis (SDS-PAGE), the lane M was the molecular weight standard, and the lane G-Fc was the target protein.

Figure 2: SDS-PAGE detection of NiV41 IgG, 41-4IgG, 41-6IgG and 41-9IgG antibodies after purification. Lanes NiV41, 41-4, 41-6, 41-9 are the target proteins.

Figure 3: Binding of NiV41 IgG, 41-4 IgG, 41-6 IgG to 41-9 IgG and Nipah virus envelope protein determined by ELISA. The EC50 of the binding concentration of NiV41 IgG and antigenic protein is 1.8 μg/ml, the binding concentration of 41-4 IgG and antigenic protein EC50 is 0.21 μg/ml, the binding concentration of 41-6 IgG and antigenic protein EC50 is 0.1 μg/ml, 41-9 IgG The EC50 concentration of the antigenic protein is 0.06μg/ml.

Figure 4: NiV41 IgG, 41-4IgG, 41-6IgG and 41-9IgG neutralization experiment of Nipah pseudovirus. The neutralizing activity IC50 of NiV41 IgG and Nipah pseudovirus is 0.01 μg/ml, the neutralizing activity IC50 of 41-4 IgG and Nipah pseudovirus is 0.005 μg/ml, and the neutralization activity of 41-6 IgG and Nipah pseudovirus The neutralizing activity IC50 of 41-9 IgG and Nipah pseudovirus was 0.002 μg/ml.

Figure 5: NiV41 IgG, 41-4IgG, 41-6IgG and 41-9IgG neutralize live Nipah virus experiment. The neutralizing activity IC50 of NiV41 IgG and Nipah live virus was 0.33 μg/ml, the neutralizing activity IC50 of 41-4 IgG and Nipah live virus was 0.136 μg/ml, and the neutralization activity of 41-6 IgG neutralized Nipah live virus The neutralizing activity IC50 of 41-9 IgG and Nipah live virus was 0.059 μg/ml.

Figure 6: 41-6 interacts with the antigenic protein NiV-G. Antigen-antibody complex structure prediction was carried out by Alpha Fold2 prediction software, and antibody 41-6 mainly participated in the interaction with antigen through the CDR3 region of the heavy chain.

Figure 7: NiV41 IgG, 41-6 IgG animal protection experiment. NiV41 IgG can provide complete therapeutic protection to infected animals, 41-6 IgG can provide complete preventive protection to infected animals, and the negative control is PBS.

Detailed ways

Below in conjunction with embodiment and accompanying drawing, the present invention is described in detail, and following embodiment is to carry out under the premise of technical solution of the present invention, has provided detailed implementation and specific operation process, but protection scope of the present invention is not limited to Examples described below.

Embodiment 1: express and purify Nipah virus envelope protein

According to the gene sequence of Nipah virus (GenBank NC_002728.1), the ectodomain (amino acid 188-602) of its envelope protein was spliced with the Fc gene sequence of IgG1 type, and the spliced gene was connected and transformed with the vector pSecTag2A to construct the vector pSecTag2A-G-Fc. One day before transfection, 40 mL of 293F cells (control cell density: 5-×10 ⁵ cells/ml) were inoculated in a 125 mL suspension cell culture flask. Dilute 40 μg of the plasmid (pSecTag2A-G-Fc) in 4 mL of DPBS buffer solution and mix gently, then dilute 120 μL of PEI (Polyethyleneimine) in the culture medium and mix gently. They were added dropwise to the cells after 20 min incubation at room temperature. The cells were placed in a suspension incubator at 125 rpm at 37°C for suspension culture.

After 144 hours, the culture supernatant was collected, and the expression of the antigen protein was detected by Western Blot using anti-Fc as the primary antibody.

After detecting the expression of G-Fc, expand the scale of cell culture and transfection to express G-Fc protein in large quantities. Collect the supernatant of the culture medium, purify the target protein with protein A filler, and then replace the buffer with an ultrafiltration centrifuge tube with a molecular weight cut-off of 10kDa, and verify its purity by SDS-PAGE. The results are shown in Figure 1.

Example 2: Construction and screening of phage display library

Using the constructed phage library, the antigens expressed by mammalian cells were screened. After the purified antigen was incubated overnight at 4°C in a 96-well plate, it was panned with a phage library, and the specific phage was captured by the antigen, washed with PBS+0.05% Tween-20, and after four rounds of screening, the obtained The enriched clone was named NiV41.

For the RNA from a healthy and non-immune donor, the gene encoding the light chain fragment of the antibody was obtained by PCR and amplified in large quantities. On the basis of the antibody gene of the parental clone NiV41, a new light chain shuffled fragment was obtained by enzyme digestion and connection. The affinity maturation library was screened again with G-Fc protein to obtain enriched clones, which were named 41-4, 41-6 and 41-9, respectively.

Example 3: Expression and purification of NiV41 IgG, 41-4IgG, 41-6IgG and 41-9IgG

The heavy and light chains of NiV41 Fab, 41-4Fab, 41-6Fab and 41-9Fab were constructed into the IgG1 expression vector pivottro2-neo-mcs (commercially available), and 293F cells were transfected with PEI for expression, and the expression supernatant was expressed with protein Filler A was purified, and then the buffer was replaced by ultrafiltration with an ultrafiltration centrifuge tube with a molecular weight cut-off of 30kDa, and its purity was verified by SDS-PAGE. The results are shown in Figure 2.

Embodiment 4: ELISA measures the combination of NiV41 IgG, 41-4IgG, 41-6IgG and 41-9IgG and envelope protein

Envelope protein (4 μg/mL) was coated on the ELISA plate, incubated overnight at 4°C and blocked with PBS+3% milk at 37°C for 1 hour. Add serially diluted antibodies, incubate at 37°C for 2 hours, wash with PBST (PBS+0.05% Tween 20) four times, and then incubate at 37°C with horseradish peroxidase (HRP)-labeled mouse anti-human Fc monoclonal antibody After 1 hr, wash four times with PBST and add ABTS for detection. The binding affinity of each antibody was calculated by data analysis software Prism fitting. The results are shown in Figure 3.

Example 5: NiV41 IgG, 41-4 IgG, 41-6 IgG and 41-9 IgG antibodies neutralize Nipah pseudovirus in Vero cells.

100 μL of Vero cells (1×10 ⁵ cells/ml) were inoculated into a 96-well plate, and cultured for 14-16 hours. According to the initial antibody concentration of 100 μg/ml, three-fold serial dilution was performed, and an equal volume of Nipah virus pseudovirus was mixed with the antibody diluent, and incubated at 37°C for 1 hour. Discard the cell culture medium in the 96-well plate, infect the cells with the virus and antibody mixture, and culture at 37°C for 24 hours. The next day, the fluorescence of the cells was observed under a fluorescence microscope. Through high-content scanning, the fluorescence intensity of each well plate was analyzed. The weaker the fluorescence, the fewer cells were infected by the pseudovirus, that is, the more pseudoviruses were neutralized by the antibody. The half inhibitory activity (IC50) of each antibody was calculated by the data analysis software Prism. The results are shown in Figure 4.

Example 6: Antibodies NiV41 IgG, 41-4 IgG, 41-6 IgG and 41-9 IgG neutralize live Nipah virus.

0.5 mL of Vero cells (1×10 ⁵ cells/ml) were inoculated into a 24-well plate, and cultured for 14-16 hours. According to the initial antibody concentration of 100ug/ml, three-fold serial dilution was performed, and an equal volume of live Nipah virus was mixed with the antibody diluent, and incubated at 37°C for 1 hour. Discard the cell culture medium in the 24-well plate, infect the cells with the virus and antibody mixture, and culture at 37°C for 1 hour. Discard the virus-antibody mixture in the cell well plate, add a medium containing carboxymethylcellulose, and culture at 37° C. for 4 to 5 days. After the cell plate was inactivated, the formation and number of plaques were observed and counted by staining with crystal violet. Fewer plaques indicate fewer virus-infected cells, that is, more pseudoviruses neutralized by antibodies. The half inhibitory activity (IC50) of each antibody was calculated by the data analysis software Prism. The results are shown in Figure 5.

Example 7: Prediction of the interaction interface between antibody 41-6 and antigen

The sequence of the Fab fragment of the antibody and the antigen sequence are written into the text, and the structure of the antigen-antibody complex is predicted in the form of a polymer through the Alpha Fold2 database. For the predicted structure, the structure with the highest score is selected for analysis. The interaction interface of the complex was analyzed in the software PDBePISA. The results are shown in Figure 6.

Embodiment 8: the protection of antibody NiV41 IgG, 41-6 IgG to the hamster that has been infected with Nipah virus

Four-five-week-old female hamsters were inoculated with a certain dose of Bengal Nipah strain (NiV _B ) by intraperitoneal injection, and 6 hours after infection, each experimental animal was injected with 300 μg of IgG antibody by intraperitoneal injection For NiV41, PBS was selected as the negative control, and the physical condition of the hamsters was observed every day and the survival conditions were recorded;

Four-five-week-old female hamsters were injected intraperitoneally, and each experimental animal was given 1 mg of IgG antibody 41-6, and PBS was used as a negative control. One day after the prophylaxis, a certain dose of Nipah virus of Malaysian origin (NiV _M ) was inoculated. The physical condition of the hamsters was observed every day and the survival conditions were recorded.

The above experiments were carried out in a biosafety level 4 laboratory. The experimental results are shown in Figure 7.

Those skilled in the art can better understand and master the present invention with the help of the examples. However, the scope of protection and claims of the present invention is not limited to the examples provided. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Claims (10)

1. An antibody heavy chain variable region directed against nipah virus G protein, characterized by: the heavy chain variable region has three complementarity determining regions CDRs of HCDR1, HCDR2 and HCDR3, the HCDR1 comprising the amino acid sequence shown in SEQ ID No. 1; the HCDR2 comprises an amino acid sequence shown as SEQ ID No. 2; the HCDR3 comprises an amino acid sequence as shown in SEQ ID No. 3.

2. The antibody heavy chain variable region against nipah virus G protein as set forth in claim 1, wherein: the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID No. 4.

3. A nipah virus antibody comprising the antibody heavy chain variable region of claim 1 or 2 directed against a nipah virus G protein.

4. The nipah virus antibody of claim 3 having a light chain, wherein: the variable region of the light chain has three complementarity determining regions CDRs, LCDR1, LCDR2 and LCDR3, selected from the group consisting of:

the LCDR1 comprises an amino acid sequence shown as SEQ ID No. 5; the LCDR2 comprises an amino acid sequence shown as SEQ ID No. 6; the LCDR3 comprises an amino acid sequence shown as SEQ ID No. 7; or (b)

The LCDR1 comprises an amino acid sequence shown as SEQ ID No. 9; the LCDR2 comprises an amino acid sequence shown as SEQ ID No. 10; the LCDR3 comprises an amino acid sequence shown as SEQ ID No. 11; or (b)

The LCDR1 comprises an amino acid sequence shown as SEQ ID No. 13; the LCDR2 comprises an amino acid sequence shown as SEQ ID No. 14; the LCDR3 comprises an amino acid sequence shown as SEQ ID No. 15; or (b)

The LCDR1 comprises an amino acid sequence shown as SEQ ID No. 17; the LCDR2 comprises an amino acid sequence shown as SEQ ID No. 18; the LCDR3 comprises an amino acid sequence as shown in SEQ ID No. 19.

5. The nipah virus antibody of claim 4, wherein: the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID No.8, SEQ ID No.12, SEQ ID No.16, or SEQ ID No. 20.

6. An antigen binding fragment comprising the antibody heavy chain variable region of claim 1 or 2 against nipah virus G protein.

7. A bispecific antibody comprising the antibody heavy chain variable region of claim 1 or 2 against nipah virus G protein, or the nipah virus antibody of any one of claims 3-5.

8. Use of the heavy chain variable region of an antibody against nipah virus G protein according to claim 1 or 2 for the preparation of a prophylactic and therapeutic drug against nipah virus, a detection probe, a fusion polypeptide, a fusion protein, an immunoconjugate, a genetically engineered host cell.

9. Use of the nipah virus antibodies of claim 3 or 4 or 5 for the preparation of prophylactic and therapeutic drugs, detection probes, fusion polypeptides, fusion proteins, immunoconjugates, genetically engineered host cells against nipah virus.

10. Use of the antigen binding fragment of claim 6 for the preparation of a prophylactic and therapeutic drug against nipah virus, a detection probe, a fusion polypeptide, a fusion protein, an immunoconjugate, a genetically engineered host cell.