C-type nano antibody for neutralizing Hunipah virus and application of C-type nano antibody

Abstract

The invention discloses a C-type nano antibody for neutralizing Henipah virus and application, and provides two C-type nano antibodies which can be specifically combined with Henipah virus fusion protein with high affinity, and have efficient neutralizing activity on Nipah pseudovirus and Hendea pseudovirus which take VSV as a skeleton. In addition, a bivalent bispecific nano antibody 2A1-3-8-HLE (a VHH targeting serum albumin is fused at the C terminal of the 2A1-3-8 to prolong the plasma half-life period of the 2A1-3-8) completely protects the hamster infected with the Nipah virus from death. The two C-type nano antibody targeted epitopes are a hidden cavity and crack formed by two adjacent protomers on the surface of the fusion protein, and the epitopes are different from the previously reported antibody epitopes. Based on the understanding of the structure, the biological activity of 2A1-3-8 is enhanced through rational design, and 1A1-3-22 has broad spectrum. In conclusion, the two C-type nano antibodies are expected to become novel specific antibody drugs for preventing and treating the Henipah virus, and more choices are provided for prevention and control of the Henipah virus.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical engineering, and particularly relates to a C-type nanobody for neutralizing henipavirus and its application. Background Art

[0002] Nipah virus (NiV) and Hendra virus (HeV) are two prototype pathogens of the genus Henipavirus in the family Paramyxoviridae. Both are biosafety level 4 (BSL-4) viruses, which can cause severe viral diseases and lead to a relatively high fatality rate (40-75%). NiV and HeV were discovered in Malaysia and Australia in 1998 and 1994 respectively. Their natural hosts are fruit bats of the family Pteropodidae. Along with the extensive geographical distribution of fruit bats, nowadays, the two viruses have been prevalent on a large scale and frequently in regions such as South Asia, Southeast Asia and Australia. Compared with Hendra virus, Nipah virus is more lethal and has a wider distribution. Nipah virus is divided into two strains: the Malaysian strain (NiV M ) and the Bangladesh strain (NiV B ). The former mainly prevails in Southeast Asia through intermediate hosts such as pigs and horses, while the latter is mainly transmitted to humans through food contaminated by fruit bats, and human-to-human transmission has been found.

[0003] NiV and HeV are single-stranded negative-sense RNA viruses, and there are two envelope proteins on the virus surface: the receptor-binding protein (RBP or G protein) and the fusion protein (F). RBP is a type II transmembrane protein, and its head domain contains a receptor-binding domain that can bind to the receptors Ephrin B2 or Ephrin B3. F is a type I membrane fusion protein, mainly responsible for mediating the fusion of the virus membrane and the cell membrane. During the process of the virus entering the host cell, first, RBP binds to Ephrin B2 or Ephrin B3 on the cell surface, and then RBP interacts with F, thereby triggering a series of conformational changes in F, changing from the pre-fusion state to the post-fusion state, and finally forming a six-helix bundle inserted into the cell membrane, thereby mediating the fusion of the virus membrane and the cell membrane and completing the virus invasion process. Thus, both RBP and F can be used as neutralization targets of the virus. Blocking the binding of RBP to the receptor, preventing the conformational change process of F, or interfering with the interaction between RBP and F can all prevent the virus from entering the cell. Currently, there are no approved drugs or vaccines for human treatment. Antibodies targeting the F protein are mainly divided into three epitopes: the top, the side, and the bottom. However, there are no antibodies targeting the epitopes near a conserved cavity, a semi-conserved pocket, and the heptad repeat region A. Therefore, these epitopes may be potential neutralization targets.

[0004] Compared with traditional full-length antibodies, nanobodies in various forms such as V-type nanobodies (VH) derived from the variable regions of antibodies, VHH from camels, or VNAR from sharks have the advantages of small size, easy expression and production, high tissue permeability, and high stability. Our previous research developed a C-type nanobody scaffold by modifying the scaffold of the IgG1 constant region 2 (CH2) domain. Compared with the above-mentioned other forms of nanobodies, C-type nanobodies are derived from the human genome and thus do not require humanization. In addition, it is itself part of the IgG1 Fc fragment and thus has some binding sites for Fc receptors, which may enable C-type nanobodies to have a longer half-life and higher stability. Phage display technology is a fast, convenient, and low-cost way to obtain various forms of antibodies. The purpose of the present invention is to construct a phage display library based on C-type nanobodies by combining the C-type nanobody scaffold with phage display technology to quickly obtain nanobodies with high binding activity and neutralizing activity for dealing with Nipah virus infection, thereby making up for the deficiencies of the current single form and low activity of antibodies. Summary of the Invention

[0005] In view of the problems in the prior art, the present invention provides a C-type nanobody for neutralizing Henipavirus and its application, which targets the Henipavirus fusion protein (also referred to as F or F protein in the present invention).

[0006] In the present invention, from the phage surface display libraries with the scaffolds of CH2-m01 and CH2-m02 modified from the CH2 domain of human IgG1, the fusion protein of Nipah virus Malaysian strain (NiV M -F), the fusion protein of Nipah virus Bangladesh strain (NiV B-F), Hendra virus fusion protein (HeV-F) as the antigen, after sequential screening and subsequent three rounds of affinity maturation, finally obtained the C-type single-domain antibodies 2A1-3-8 that can broadly neutralize Nipah virus and Hendra virus, and the C-type single-domain antibody 1A1-3-22 that specifically neutralizes Nipah virus. We expressed and purified these two clones in Escherichia coli, and then characterized the in vitro biological activities of these two clones. The binding activities (or affinities) of 2A1-3-8 and 1A1-3-22 were identified by enzyme-linked immunosorbent assay and biolayer interferometry, and then the neutralization activities were tested on pseudoviruses and authentic viruses based on the VSV backbone. By resolving the high-resolution structure of the complex of 2A1-3-8 or 1A1-3-22 with NiV-F by cryo-electron microscopy, it was found that these two nanobodies bind to similar epitopes of the antigen, namely domain I, III of one protomer, and domain I, II of the adjacent protomer. Binding to the conserved cavity formed by the two protomers and the crevice around the cavity is the most prominent feature of these two nanobodies. The most promising C-type nanobody 2A1-3-8 was constructed into a bispecific bivalent nanobody 2A1-3-8-HLE by fusing a VHH targeting serum albumin at its C-terminus, which can be based on pharmacokinetic parameters 50 of NiV M to rationally design the dosing window and dosing interval under the challenge of, and play a complete protective role on hamsters. The above experiments indicate that these two C-type nanobodies can be used for the prevention and treatment of henipavirus.

[0007] Analysis of the antigen-antibody complex structure showed that two amino acids Y59 and F60 on 2A1-3-8 Loop 2 play very important roles in the biological activity and broad-spectrum nature of 2A1-3-8, probably because the aromatic rings of the side chains of these two amino acids can fill the cavity epitope on the antigen surface. Therefore, we carried out single-point mutations and combinatorial mutations of three aromatic amino acids (the side chains are F, Y, W in ascending order) at these two sites and found certain rules: the larger the side chain of the aromatic amino acid at position 59 and the smaller the side chain of the aromatic amino acid at position 60, the better the neutralization activity of the nanobody. Since Loop 2 of 2A1-3-8 and Loop1 of 1A1-3-22 are spatially close, we replaced 1A1-3-22-Loop1 with 2A1-3-8-Loop 2 and obtained a chimeric antibody 1A1-3-22Loop 2-grafting, and found that this chimeric antibody has the ability to bind to HeV-F and the neutralization activity against HeV pseudovirus has been increased by about 7 times.

[0008] 2A1-3-8 and its mutants have three Loop regions, and the amino acid sequences of the three Loop regions, Loop 1, Loop 2, and Loop 3, are shown in SEQ ID No.1, SEQ ID No.2-10, and SEQ ID No.11, respectively.

[0009] 1A1-3-22 has three Loop regions, and the amino acid sequences of the three Loop regions, Loop 1, Loop 2, and Loop 3, are shown in SEQ ID No.12, SEQ ID No.13, and SEQ ID No.14, respectively.

[0010] The amino acid sequences of 2A1-3-8 and its mutants are shown in SEQ ID No.15-23.

[0011] The amino acid sequences of 1A1-3-22 and its chimeras are shown in SEQ ID No.24-25.

[0012] To extend the plasma half-life of 2A1-3-8, a VHH targeting serum albumin was fused to its C-terminus to construct a bivalent bispecific nanobody (2A1-3-8-HLE), and its amino acid sequence is shown in SEQ ID No.26.

[0013] The present invention also discloses the application of the above-mentioned C-type single-domain antibodies that neutralize henipavirus in the preparation of preventive and therapeutic drugs, detection probes, fusion polypeptides, fusion proteins (such as tandem forms of bivalent and multivalent nanobodies, tandem forms of bispecific and multispecific nanobodies), antibody cocktail therapies, and conjugated antibodies against henipavirus. The C-type single-domain antibodies that neutralize henipavirus of the present invention can be used in the preparation of nasal drops, nasal sprays, nebulized drugs, intramuscular injection preparations, and intravenous injection preparations related to the prevention and treatment of henipavirus.

[0014] The term "C-type single-domain antibody" used in the present invention refers to a class of antibodies with the constant region CH2 domain of an antibody as the backbone. Compared with full-length monoclonal antibodies, single-domain antibodies have better tissue permeability and can preferentially bind to antigen epitopes with steric hindrance effects in space.

[0015] The beneficial effects of the present invention are as follows: The two C-type nanobodies provided can bind to Hendra virus F with high affinity and neutralize Hendra pseudovirus and live virus. Their neutralization mechanisms are both to lock F in the pre-fusion conformation, prevent its transformation into the post-fusion conformation, thereby blocking the membrane fusion process and further inhibiting virus entry into cells. In addition, the nanobody 2A1-3-8-HLE with an extended half-life can completely protect hamsters from death caused by Nipah virus infection, which is the first application of nanobodies in the treatment of infections with category 4 viruses. In addition, the antigenic epitopes targeted by these two C-type nanobodies are a new epitope that has never been reported before, which can provide a structural basis for future epitope-based vaccines or antibody-based vaccine design. Based on the understanding of the structure, we enhanced the neutralization activity of 2A1-3-8 through point mutations of aromatic amino acids and also made 1A1-3-22 broad-spectrum through Loop grafting. Compared with full-length monoclonal antibodies (molecular weight ~150 kD), the C-type nanobodies of the present invention have a smaller molecular weight (12 - 15 kD), better tissue permeability, and the ability to bind to antigenic epitopes with steric hindrance effects; they can be expressed in multiple expression systems including prokaryotic, yeast, and mammalian cells, with low production costs and short production cycles; C-type single-domain antibodies have a wide range of administration routes and can be administered by means such as nasal drops, nasal spray, aerosol spray, nebulized inhalation, intramuscular injection, subcutaneous injection, intravenous injection, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Detection of NiV-F using SDS-PAGE M -F (left), NiV B -F (middle), and HeV-F (right) for the expression and purification of three Hendra virus F proteins.

[0017] Figure 2 : Expression and purification of C-type single-domain antibodies 2A1-3-8 (left) and 1A1-3-22 (right).

[0018] Figure 3 : Binding activities of 2A1-3-8 and 1A1-3-22 to three Hendra virus F proteins NiV M -F, NiV B -F, and HeV-F detected by ELISA, with the published positive antibody h5B3.1 used as a positive control.

[0019] Figure 4 : Neutralization activities of 2A1-3-8 and 1A1-3-22 against NiV M and pseudovirus, with the published positive antibody h5B3.1 used as a positive control.

[0020] Figure 5: Neutralize NiV in 2A1-3-8 and 1A1-3-22 M , NiV B , the activities of the true viruses of HeV, where the published positive antibody h5B3.1 was used as a positive control and high-concentration C3 was used as a negative control.

[0021] Figure 6 : 2A1-3-8 or 1A1-3-22 inhibits syncytium formation, and qualitative analysis was performed by the expression of GFP (green fluorescent protein), where the published positive antibody h5B3.1 was used as a positive control and a single high-concentration C3 was used as a negative control.

[0022] Figure 7 : 2A1-3-8 or 1A1-3-22 inhibits syncytium formation, and quantitative analysis was performed by the activity of Renilla luciferase, where the published positive antibody h5B3.1 was used as a positive control and a single high-concentration C3 was used as a negative control.

[0023] Figure 8 : NiV M -F and the high-resolution cryo-EM structure of the complex formed with 2A1-3-8 (a) or 1A1-3-22 (b).

[0024] Figure 9 : The pharmacokinetic curves of 2A1-3-8 (a) and 2A1-3-8-HLE (b) in hamsters (4 in each group).

[0025] Figure 10 : 2A1-3-8-HLE protects hamsters from NiV M The survival curve (a) and body weight change curve (b) of live virus infection.

[0026] Figure 11 : The activities of 2A1-3-8 and its point mutants in neutralizing Nipah pseudovirus and Hendra pseudovirus.

[0027] Figure 12 : The binding activities of 1A1-3-22 and its chimera 1A1-3-22Loop 2-grafting to the F proteins of three Henipaviruses detected by ELISA.

[0028] Figure 13 : The activities of 1A1-3-22 and its chimera 1A1-3-22Loop 2-grafting in neutralizing Nipah pseudovirus and Hendra pseudovirus. Detailed implementation methods

[0029] The present invention will be described in detail below in conjunction with embodiments and the accompanying drawings. The following embodiments are implemented on the premise of the technical solution of the present invention, and the detailed implementation manners and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0030] Example 1: Expression and purification of three soluble henipavirus fusion proteins

[0031] The extracellular domains (amino acids 1 - 487) of codon-optimized NiV M -F (GenBank: AY029767.1), NiV B -F (GenBank: AY988601.1) and HeV-F (GenBank: AF017149.3) were fused with the C-terminus of the GCN4 trimer motif and a 6×HIS tag, and the F was stabilized in the pre-fusion conformation by the following point mutations: NiV M -F (L104C / I114C / L172F / S191P), NiV B -F (L104C / I114C / L172F), HeV-F (L104C / V114C / L172F). Finally, it was constructed into the pCAGGS vector through the restriction enzyme sites EcoR I and Not I. One day before transfection, 40 mL of 293F cells (control cell density about 1×10 6 cells / mL) were inoculated into a 125 mL suspension cell culture flask and cultured overnight. 40 μg of plasmid was diluted in 4 mL of DPBS and mixed well by inverting up and down. Then 120 μL of PEI (polyethylenimine) was diluted in DPBS and mixed well by inverting up and down. After incubating at room temperature for 20 - 30 minutes, the mixture was added dropwise to the cells. The cells were placed in a suspension culture incubator and cultured in suspension at 130 rpm / min, 37 °C, 8% CO2 for 5 - 6 days. During this period, 50 μL of samples were taken every day, and the protein expression was detected by Western Blot using Alkaline phosphatase conjugated-anti-6×HIS.

[0032] After detecting the expression of the target protein, the cell culture and transfection scale were expanded to express the above three henipavirus fusion proteins in large quantities. The culture medium supernatant was collected, and the target protein was purified with Ni filler, and then the buffer was ultrafiltered and replaced with an ultrafiltration centrifugal tube with a molecular weight cut-off of 30 kD. After final concentration, its purity was verified by SDS-PAGE, as shown in Figure 1 .

[0033] Example 2: Construction and screening of a phage display library

[0034] Using human CH2 as the backbone, a phage display library was constructed according to the existing literature (Gong R., et al., PLoS One, 2012, 7: e42288). Sequential screening was performed with three eukaryotic-expressed F proteins: After biotinylated antigen was conjugated to magnetic beads, screening was carried out using the phage display library. During the screening process, phages specific to the F protein could be captured by the antigen, and phages that could not bind to the F protein were washed away. After four rounds of screening, candidate clones 2A1 and 1A1 were obtained. Random mutations were introduced into the sequences of these two parental clones by error-prone PCR, and then sequential screening was performed again with the F protein, which was one round of affinity maturation. After three rounds of affinity maturation, the final candidate clones 2A1-3-8 and 1A1-3-22 were obtained.

[0035] Example 3: Expression and purification of 2A1-3-8 and 1A1-3-22

[0036] The expression and purification of two C-type nanobodies were carried out according to the existing literature (Gong R., et al., Methods Mol Biol., 2012, 899: 85-102). The prokaryotic expression vectors of 2A1-3-8 and 1A1-3-22 were transformed into E. coli HB2151. Single colonies were picked and inoculated into SB medium containing 100 μg / mL ampicillin (30 g tryptone, 20 g yeast extract, and 10 g MOPS in 1 L medium, pH adjusted to 7.0 with NaOH). When the OD 600 reached 0.7 - 1.0, IPTG was added to a final concentration of 200 μg / ml, and induction expression was carried out at 37 °C and 220 rpm for 14 - 16 hours. The cells were collected by centrifugation at 4 °C, 6000 rpm for 15 min, the culture medium was discarded, the precipitate was resuspended in PBS (pH 7.0), and then treated with polymyxin B for 1 hour and the supernatant was collected by centrifugation. Purification was carried out using Ni-NTA resin, and then concentrated and replaced with an ultrafiltration centrifugal tube with a molecular weight cut-off of 3 kD. The expression and purification of the obtained 2A1-3-8 and 1A1-3-22 are shown in Figure 2 .

[0037] Example 4: ELISA determination of the binding of 2A1-3-8 and 1A1-3-22 to henipavirus F

[0038] NiV M -F, NiV B-F and HeV-F (4 μg / mL) were coated on ELISA plates and incubated overnight at 4 °C, then blocked with 3% milk at 37 °C for 1.5 hours. Gradient-diluted 2A1-3-8 and 1A1-3-22 were added, incubated at 37 °C for 1.5 hours, washed 5 times with PBST (PBS + 0.05% Tween 20), then incubated with HRP-anti-FLAG monoclonal antibody at 37 °C for 1 hour, washed 5 times with PBST, and then ABTS was added for color development. The binding activities of the two nanobodies to different F proteins were calculated by fitting with the data analysis software GraphPad Prism, and the results are shown in Figure 3 . 2A1-3-8 binds to NiV M -F, NiV B -F and the half-maximal effective concentration (EC 50 ) of HeV-F were 0.012 μg / mL, 0.005 μg / mL, and 0.008 μg / mL respectively; 1A1-3-22 binds to NiV M -F, NiV B -F's EC 50 values were 0.013 μg / mL and 0.004 μg / mL respectively and did not bind to HeV-F.

[0039] Example 5: Neutralization of Nipah pseudovirus by 2A1-3-8 and 1A1-3-22 antibodies in Vero cells

[0040] 100 μL of Vero cells (2×10 4 cells / well) were seeded in 96-well plates and cultured for 14 - 16 hours. After gradient-diluting the antibodies, they were mixed with an equal volume of Nipah virus pseudovirus and antibody diluent and incubated at 37 °C for 1 hour. The cell culture medium in the 96-well plates was discarded, and the cells were infected with the virus and antibody mixture and incubated statically at 37 °C for 24 hours. The next day, through high-content scanning, the number of eGFP expressed in the cells at different antibody dilutions was counted, and the inhibition rate was calculated based on the virus wells without antibodies. The half-maximal inhibitory activity (IC 50 ) of each antibody was calculated by the data analysis software GraphPad Prism, and the results are shown in Figure 4 . The half-maximal inhibitory concentration (IC M ) of 2A1-3-8 and 1A1-3-22 against NiV 50 pseudovirus were 0.0002 μg / mL and 0.0043 μg / mL respectively

[0041] Example 6: Neutralization of live Nipah virus by 2A1-3-8 and 1A1-3-22

[0042] 0.5 mL of Vero E6 cells (1.4×10 5(cells / well) were seeded into 24-well plates and cultured for 14 - 16 h. The antibodies were serially diluted five-fold and then mixed with an equal volume of three Hendra live viruses and antibody diluent, and incubated at 37 °C for 1 h. The cell culture medium in the 24-well plates was discarded, and the cells were infected with the virus and antibody mixture and incubated statically at 37 °C for 1 h. The virus-antibody mixture in the cell wells was discarded, and a medium containing carboxymethyl cellulose was added, and the cells were incubated statically at 37 °C for 4 - 5 days. After inactivating the cell plates, crystal violet staining was performed to observe and count the formation and number of plaques. The fewer the plaques, the fewer the cells infected by the virus, that is, the more pseudoviruses neutralized by the antibody. The half inhibitory activity (IC 50 ) of each antibody was calculated using the data analysis software GraphPad Prism, and the results are shown in Figure 5 . 2A1-3-8 neutralized NiV M , NiV B and the IC 50 values of HeV live virus were 0.00039 μg / mL, 0.0168 μg / mL, and 0.0028 μg / mL, respectively; 1A1-3-22 neutralized NiV M , NiV B and the IC 50 values of HeV live virus were 0.2952 μg / mL, 1.1685 μg / mL, and 19.9342 μg / mL, respectively.

[0043] Example 7: 2A1-3-8 and 1A1-3-22 prevent virus entry into cells by inhibiting the formation of Nipah virus syncytia

[0044] One day before the experiment, receptor-negative cells PK-13 were seeded in 6-well plates at 6×10 5 cells / well; meanwhile, receptor-positive cells 293T were seeded in 6-well plates at 1.2×10 6 cells / well. On the second day, NiV M -F, NiV M -RBP and DSP 1-7 were transfected into PK-13, and DSP 8-11 was transfected into 293T. On the third day, after detaching PK-13, it was mixed with serially diluted 2A1-3-8 and 1A1-3-22 at an equal volume (75 μL + 75 μL) and incubated at 37 °C for 1 h. Subsequently, after serially diluting the antibodies five-fold, they were mixed with 293T at an equal volume (150 μL + 150 μL), and the antibody-cell mixture was added to 48-well plates and cultured for 24 h. Subsequently, qualitative analysis was performed by observing the expression of GFP under a fluorescence microscope (see Figure 6 ), and quantitative analysis was performed by detecting the activity of luciferase (see Figure 7 ).

[0045] Example 8: 2A1-3-8 and 1A1-3-22 with NiV M -F trimer complex structure analysis

[0046] 2A1-3-8 and 1A1-3-22 with NiV M After 2A1-3-8 and 1A1-3-22 were incubated with NiV Figure 8 -F to form a complex at a molar ratio of 6:1, the complex was separated by size-exclusion chromatography, and a cryo-EM sample was prepared. Preliminary screening was carried out using a 200 kV cryo-electron microscope. Copper grids with samples meeting the requirements were then used to collect single-particle images through a 300 kV cryo-electron microscope. After a series of processes such as 2D classification, 3D averaging, and model building, the structure of the complex was analyzed. is the high-resolution complex structure of 2A1-3-8 and 1A1-3-22 with NiV

[0047] -F. The results showed that both 2A1-3-8 and 1A1-3-22 bind in the cavities and crevices formed by two adjacent protomers, interacting with domain I, III of one protomer, and domain I, II of the adjacent protomer, suggesting that this region can be a potential target for broad-spectrum vaccines and antibodies.

[0048] Figure 9 Example 9: Pharmacokinetic analysis of 2A1-3-8 and 2A1-3-8-HLE To determine the pharmacokinetics of 2A1-3-8 and 2A1-3-8-HLE in hamsters, female hamsters aged 4-6 weeks (4 per group) were selected for the study. 2A1-3-8 and 2A1-3-8-HLE expressed in E. coli were endotoxin-free and aliquoted. Each group of hamsters received an intraperitoneal injection of 0.5 mg (about 5 mg / kg) of 2A1-3-8 or 2A1-3-8-HLE. Blood was collected from the retro-orbital vein at 0.2, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, 144, 192, and 240 hours after dosing. First, a standard curve and regression equation for 2A1-3-8 and 2A1-3-8-HLE binding to NiV

[0049] -F were obtained by ELISA. Subsequently, sera at different time points were serially diluted, and the antibody concentration in the serum of each hamster at each time point was calculated using the regression equation. The pharmacokinetic curves of each hamster were plotted using GraphPad Prism ( 1 / 2 ). The non-compartmental model in Phoenix WinNonlin 8.1 software was used to calculate the pharmacokinetic parameters, and the plasma half-life (t 1 / 2 ) of 2A1-3-8 was 2.32 hours, and that of 2A1-3-8-HLE was 49.90 hours.

[0050] Example 10: Protection effect of 2A1-3-8-HLE against NiV M live virus

[0050] Twenty-four female hamsters aged 4-6 weeks were randomly divided into groups of 6 each. At the 0th hour (i.e., the virus challenge time point), 1000 LD 50 of NiV M live virus was injected intraperitoneally. Three of the groups were intraperitoneally injected with 1 mL of 20 mg / mL 2A1-3-8-HLE, 10 mg / mL 2A1-3-8-HLE, and PBS respectively at 2 hours, 1 day, and 3 days after infection. Another group was intraperitoneally injected with 1 mL of 20 mg / mL 2A1-3-8-HLE at 1 day, 3 days, and 5 days after infection. The survival of the hamsters was observed daily, and the weight changes were recorded. The survival curve and weight change curve were plotted using GraphPad Prism. See Figure 10 .

[0051] Example 11: Construction and biological activity of 2A1-3-8 and its point mutants

[0052] The 2A1-3-8 Loop 2 obtained by screening contains two point mutations, Y59 and F60. Single-site mutations and double-site combination mutations of three aromatic amino acids (F, Y, W) were performed on the amino acids at these two sites, and a total of 9 mutants including 2A1-3-8 were obtained: 2A1-3-8 (also referred to as 2A1-3-8YF hereinafter), 2A1-3-8FF (i.e., 2A1-3-8Y59F), 2A1-3-8FY (i.e., 2A1-3-8Y59F / F60Y), 2A1-3-8FW (i.e., 2A1-3-8Y59F / F60W), 2A1-3-8YY (i.e., 2A1-3-8F60Y), 2A1-3-8YW (i.e., 2A1-3-8F60W), 2A1-3-8WF (i.e., 2A1-3-8Y59W), 2A1-3-8WY (i.e., 2A1-3-8Y59W / F60Y), 2A1-3-8WW (i.e., 2A1-3-8Y59W / F60W). The above mutants were expressed in E. coli, and the protein expression method was referred to Example 3. The obtained point mutant proteins were subjected to neutralization experiments with Nipah pseudovirus and Hendra pseudovirus, and the detection method was referred to Example 5. The detection results are as Figure 11 shown. The IC 50 of these point mutant antibodies against Nipah pseudovirus was between 0.2-0.5 ng / mL, and the IC 50 against Hendra pseudovirus was between 3-40 ng / mL, and all showed the following rule: the larger the side chain of the aromatic amino acid at position 59 and the smaller the side chain of the aromatic amino acid at position 60, the better the neutralization activity of the nanobody.

[0053] Example 12: Construction and Biological Activity of 1A1-3-22 and Its Chimera

[0054] Due to their close spatial positions, the chimera constructed by replacing Loop 1 of 1A1-3-22 with 2A1-3-8 Loop 2 is 1A1-3-22 Loop 2-grafting. Protein expression of this chimera was carried out in Escherichia coli, and the protein expression method was referred to Example 3. The obtained chimeric antibody was verified for its binding activity by ELISA, and its neutralizing activity against pseudovirus was detected. The detection methods were referred to Examples 4-5 respectively. The detection results are as Figures 12 - 13 shown. At the binding level, although the binding activity of the chimera to NiV M -F and NiV B -F decreased by about 50-60 times, it possessed the binding activity to HeV-F, with an EC 50 of 0.7594 μg / mL; at the pseudovirus neutralization level, the chimera had a comparable ability to neutralize Nipah pseudovirus with 1A1-3-22, but the IC 50 for neutralizing Hendra pseudovirus increased by about 7 times compared with 1A1-3-22.

[0055] Finally, a sequence information table of all antibodies and their fragments involved in the above examples is attached.

[0056] Table 1. Sequence Information Table of Antibodies and Their Loops

[0057]

[0058]

[0059] Those skilled in the relevant art can better understand and master the present invention with the help of the examples. However, the protection scope and claims of the present invention are not limited to the provided cases. Based on the examples in the present invention, all other examples obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention.

Claims

1. A C-type nanobody that neutralizes Henipavirus, characterized in that: The C-type nanobody for neutralizing Henipavirus has a CH2 domain as a skeleton and has three Loop regions: Loop 1, Loop 2 and Loop 3; The amino acid sequences of Loop 1, Loop 2, and Loop 3 are shown in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 11, respectively; or the amino acid sequences of Loop 1, Loop 2, and Loop 3 are shown as SEQ ID No.1, SEQ ID No.3, and SEQ ID No.11, respectively; or the amino acid sequences of Loop 1, Loop 2, and Loop 3 are shown as SEQ ID No. 1, SEQ ID No. 4, and SEQ ID No. 11, respectively; or the amino acid sequences of Loop 1, Loop 2, and Loop 3 are shown as SEQ ID No.1, SEQ ID No.5, and SEQ ID No.11, respectively; or the amino acid sequences of Loop 1, Loop 2, and Loop 3 are shown as SEQ ID No. 1, SEQ ID No. 6, and SEQ ID No. 11, respectively; or the amino acid sequences of Loop 1, Loop 2, and Loop 3 are shown as SEQ ID No. 1, SEQ ID No. 7, and SEQ ID No. 11, respectively; or the amino acid sequences of Loop 1, Loop 2, and Loop 3 are shown as SEQ ID No. 1, SEQ ID No. 8, and SEQ ID No. 11, respectively; or the amino acid sequences of Loop 1, Loop 2, and Loop 3 are shown as SEQ ID No. 1, SEQ ID No. 9, and SEQ ID No. 11, respectively; or the amino acid sequences of Loop 1, Loop 2, and Loop 3 are shown as SEQ ID No.1, SEQ ID No.10, and SEQ ID No.11, respectively; or the amino acid sequences of Loop 1, Loop 2, and Loop 3 are shown as SEQ ID No.12, SEQ ID No.13, and SEQ ID No.14, respectively; Or the amino acid sequences of Loop 1, Loop 2, and Loop 3 are shown as SEQ ID No.2, SEQ ID No.13, and SEQ ID No.14, respectively.

2. The C-type nanobody for neutralizing Henipavirus according to claim 1, characterized in that: The amino acid sequence of the C-type nanobody that neutralizes Henipavirus is selected from any one of SEQ ID No.15-25.

3. Use of the C-type nanobody for neutralizing Henipavirus according to claim 1 or 2 in the preparation of preventive and therapeutic drugs, detection probes, fusion polypeptides, fusion proteins, and conjugated antibodies against Henipavirus.

4. Use of the C-type nanobody for neutralizing Henipavirus according to claim 1 or 2 in the preparation of nasal drops, nasal sprays, aerosols, and injection preparations for the prevention and treatment of Henipavirus.

5. A bivalent bispecific nanobody with an extended half-life that neutralizes Henipavirus, characterized in that The antibody comprises the amino acid sequence shown in SEQ ID No.26.

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