Anti-h5 subtype avian influenza virus nanobodies and methods of construction and use thereof

Abstract

This application relates to the field of biotechnology and discloses an anti-H5 subtype avian influenza virus nanobody with the amino acid sequence shown in SEQ ID NO: 1. Because the antigenic evolution of H5AIV poses a significant challenge to the development of highly effective and durable vaccines or antiviral drugs, the nanobody targeting conserved epitopes disclosed in this application can be used as a preventive or therapeutic agent and guide the design of universal candidate vaccines with broad and durable protection. In addition, this application also discloses the use of the anti-H5 subtype avian influenza virus nanobody, an anti-H5 subtype avian influenza virus drug, and a method for constructing the anti-H5 subtype avian influenza virus nanobody.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to nanobodies against H5 subtype avian influenza virus, their construction methods, and applications. Background Technology

[0002] Based on the phylogenetic analysis of hemagglutinin (HA), the H5 subtype of highly pathogenic avian influenza virus (HPAIV) that has broken out in poultry can be divided into 9 different evolutionary branches (branch 0-9), some of which are further subdivided into sub-branch.

[0003] Currently, the main methods for combating avian influenza virus infection are vaccination or antiviral drug administration. Based on the prevalent strains at the time, inactivated vaccines Re5, Re6, Re8, Re10, Re11, Re12, Re13, and Re14 have been produced and used to reduce the impact of H5 HPAIV on the poultry industry. However, vaccine production against emerging viruses is expected to take at least several months, and the effectiveness of antiviral drugs is limited due to the emergence of viral resistance. Therefore, antibody therapy remains an important and feasible treatment option against the virus. To date, many monoclonal antibodies (mAbs) that neutralize AIV and target ancestral H5Nx lineage viruses have been reported, but only a few antibodies are able to target circulating clades 2.3.4.4 or 2.3.2.1.

[0004] Nanobodies have been used as diagnostic and therapeutic agents due to their desirable properties such as high solubility, good thermal stability, ease of production, and small size. Nanobodies, also known as variable domains (VHHs) of heavy chain antibodies, are antigen-binding components of heavy chain antibodies (HCAbs) produced by camelids, with a molecular weight of approximately 15 kDa. Due to their long and small complementary determinant 3 (CDR3) region, nanobodies can penetrate the lumen of immunogens. This suggests that VHHs may be able to target certain epitopes that conventional antibodies cannot access; in turn, this provides valuable information for structure-based vaccine design. However, research on nanobodies targeting the H5 subtype or currently prevalent strains is limited. Furthermore, most mAbs studied in animal studies are typically administered intraperitoneally or intravenously. In contrast, equivalent amounts of antibodies or nanobodies delivered intratracheally or intranasally have been shown to reach the respiratory epithelium directly, resulting in higher survival rates and significant weight recovery compared to mAbs administered intraperitoneally or intravenously. Therefore, intratracheal or intranasal delivery of antibodies or nanobodies may enhance their neutralizing efficacy and reduce the required dosage, thereby lowering costs and increasing the feasibility of clinical applications.

[0005] HA is initially produced as a single polypeptide, which assembles into a trimer structure called HA0. During infection, host proteases cleave HA0 into its subunits HA1 and HA2. The receptor-binding site (RBS), part of subunit HA1, is located in the globular head domain of the HA molecule; it is important for attachment to the host receptor and facilitates viral entry during influenza infection. Subunit HA2, along with several residues of subunit HA1, forms the conserved stem region of HA, which enables the virus and cell membrane to fuse upon activation in the acidic environment of the body.

[0006] The paper "Research Progress in Nanobody Screening and Expression Technology" published by Zhu Guang, Wang Yichen, Song Shasha, Zhang Maishou, Wang Jiacai, et al., records commonly used nanobody screening technologies, including phage display, yeast two-hybrid technology, mRNA display, high-throughput sequencing, and mass spectrometry. In yeast two-hybrid technology, a recombinant vector containing a known protein gene sequence and a BD domain sequence, called the "decoy," needs to be constructed. A vector containing an unknown protein gene sequence and an AD domain sequence, called the "prey," needs to be constructed. If there is no interaction between the decoy and prey, BD and AD will not be activated, and the reporter gene cannot be activated and expressed. In the host cell, if the decoy and prey interact and are expressed, then BD and AD will bind to the activation sequence, and the downstream reporter gene will also be activated and expressed.

[0007] This demonstrates that antibodies obtained through screening using the yeast two-hybrid system exhibit extremely high specificity compared to screening decoys.

[0008] The problem this solution needs to solve is: how to provide a nanobody that targets the 2.3.4.4 or 2.3.2.1 cyclic evolutionary branch virus. Summary of the Invention

[0009] The purpose of this application is to provide a nanobody targeting circulatory clade 2.3.4.4 or 2.3.2.1 of the virus, to enrich the current abundance of nanobodies targeting H5 subtype circulatory clade 2.3.4.4 or 2.3.2.1.

[0010] To achieve the above objectives, this solution provides a nanobody against H5 subtype avian influenza virus, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0011] Preferably, the nucleotide sequence is as shown in SEQ ID NO: 2.

[0012] Preferably, the anti-H5 subtype avian influenza virus nanobody is designed based on the H5-Re8 virus.

[0013] In addition, this application also discloses the use of the anti-H5 subtype avian influenza virus nanobody as described above in the preparation of anti-H5 subtype avian influenza virus drugs.

[0014] Preferably, the anti-H5 subtype avian influenza virus drug is specifically a drug of the 2.3.2.1 branch and / or the 2.3.4.4 branch of the H5 subtype avian influenza virus.

[0015] Preferably, the anti-H5 subtype avian influenza virus drug is specifically an anti-H5-Re14 virus drug.

[0016] In addition, this application also discloses an anti-H5 subtype avian influenza virus drug containing the above-mentioned anti-H5 subtype avian influenza virus nanobody.

[0017] Furthermore, this application also discloses a method for constructing the aforementioned anti-H5 subtype avian influenza virus nanobody, comprising the following steps:

[0018] Step 1: Immunize camels with an inactivated or attenuated vaccine of H5 subtype avian influenza virus to induce antibody production in the camels;

[0019] Step 2: Use a yeast two-hybrid system and HA1 of H5 subtype avian influenza virus as bait to screen for antibodies and obtain target antibodies;

[0020] Step 3: Sequencing and expressing the target antibody to obtain nanobodies against H5 subtype avian influenza virus.

[0021] Preferably, step 1 specifically involves immunizing camel animals with an inactivated vaccine containing H5 subtype avian influenza virus to induce the camel animals to produce antibodies.

[0022] The beneficial effects of this application are: the antigenic evolution of H5 AIV poses a significant challenge to the development of highly effective and durable vaccines or antiviral drugs. Nanobodies targeting conserved epitopes can be used as preventive or therapeutic agents and offer the design of universal candidate vaccines with broad and durable protection. This application screened a broad-spectrum neutralizing nanobody Nb10 against H5 subtype avian influenza virus, and its good neutralizing activity was demonstrated in both in vivo and in vitro experiments. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of blue colonies growing on a QDO / X / A plate after pGBKT7-Re8-HA1 was transformed into competent cells.

[0024] Figure 2 This is a page diagram of the purified Nb10 nanobody protein;

[0025] Figure 3 The survival curve of Re6 / PR8 in mice;

[0026] Figure 4 The graph shows the body weight curve of Re6 / PR8 in mice;

[0027] Figure 5 The survival curve of Re8 / PR8 in mice;

[0028] Figure 6 The graph shows the body weight curve of Re8 / PR8 in mice;

[0029] Figure 7 The survival curve of Re10 / PR8 in mice;

[0030] Figure 8 The graph shows the body weight curve of Re10 / PR8 in mice;

[0031] Figure 9 The survival curve of Re11 / PR8 in mice;

[0032] Figure 10 The graph shows the body weight curve of Re11 / PR8 in mice;

[0033] Figure 11 The survival curve of Re14 / PR8 in mice;

[0034] Figure 12 The graph shows the body weight curve of Re14 / PR8 in mice. Detailed Implementation

[0035] The present application will be clearly and completely described below with reference to its embodiments. It should be noted that, unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0036] Example 1

[0037] Constructing a yeast nanobody library targeting H5 subtype avian influenza virus

[0038] A healthy two-year-old male Bactrian camel was vaccinated with 3 ml of the inactivated virus vaccine H5-Re8 (Harbin Weike Biotechnology Development Co., Ltd., China). This procedure was performed five times, three weeks apart. Three weeks after the last booster dose, 200 ml of blood was drawn to isolate peripheral blood mononuclear cells (PBMCs). PBMCs were isolated using density gradient separation and total RNA was extracted from them. The gene fragment encoding VHH was amplified by a two-step nested PCR. The VH–CH1–CH2 and VHH–CH2 regions were amplified using reverse transcription PCR with forward and reverse primers CALL001 and CALL002, respectively. The amplified fragment of approximately 750 bp was then purified and used as a template for the second PCR. The VHH coding sequence of approximately 400 bp was amplified by the second PCR using nested primers VHH-Forward and VHH-Reverse. Subsequently, the VHH cDNA was ligated to the linearized vector pGADT7-Rec using primers pGADT7-Rec-F and pGADT7-Rec-R. The plasmid was then introduced into competent yeast Y187 cells using lithium acetate conversion technology. After culturing at 28°C for 3 days, all cells were collected and stored at -80°C for later use, thus obtaining the Y2H library. Primers are shown in Table 1:

[0039] Table 1

[0040]

[0041] Example 2

[0042] Yeast two-hybrid system for screening nanobodies

[0043] The coding sequence of the bait protein Re8-HA1 was amplified using primers bait-HA1-F and bait-HA1-R, and then ligated to linearized pGBKT7 using primers pGBKT7-F and pGBKT7-R to construct the recombinant bait vector pGBKT7-Re8-HA1. Y2HGold competent cells were transformed using chemical transformation, and then the cells were seeded on SD medium lacking tryptophan (SD / -Trp) agar and cultured at 30°C for 3-5 days. Single colonies were picked for colony PCR verification, and sequencing confirmed the correctness of the inserted sequence. Primers used were GAL4AD-F and 3AD-R. For the screening process, newly cultured OD cells were... 600≈1.0 bait strains in the logarithmic growth phase were concentrated and mixed with 1 ml of the nanobody library yeast strain Y187 (pGADT7-preys), and cultured at 30-50 rpm for 20 hours to promote diploid formation. Cells were then plated on DDO / X / A (leucine and tryptophan deficient, SD / –Leu / –Trp, supplemented with 40 μg / ml X-alpha(α)-Gal and 200 ng / ml aureobasidin A) plates for initial screening. Figure 1 Incubate at 30℃ for 3-5 days and observe blue colony formation. Transfer the blue colonies grown on DDO / X / A plates to more stringent QDO / X / A (lacking adenine, histidine, tryptophan, and leucine; SD / –Ade / –His / –Leu / –Trp, with X-α-Gal and Staphylococcus aureus A added) agar plates and incubate at 30℃ for 3-5 days for more rigorous screening, selecting well-growing blue colonies. Perform DNA sequencing on the resulting potential positive clones. Primers are shown in Table 2.

[0044] Table 2

[0045]

[0046] A broad-spectrum neutralizing nanoantibody against H5 subtype avian influenza virus was obtained through screening, and its amino acid sequence is shown in SEQ ID NO: 1; QVQLQESGGGSVQAGGSLRLSCSASEYTRSMAWFRQVPGKEREGVAAIDTGNGNTYYPTVDGRFIISRGNAKNSVDLEMNSLTPDDTAIYYCAATQGPLWPTLGTQFSTESYNYWGQGTLVTVSS;

[0047] The nucleotide sequence encoding the above-mentioned broad-spectrum neutralizing nanobody against H5 subtype avian influenza virus is shown below (SEQ ID NO: 2):

[0048] Caagttcagttacaggaatctgggggaggctcggtgcaggctggagggtctctgagactctcctgttcagcctctgaatacactcgaagtatggcctggttccgccaggttccagggaaggagcgcgagggggtcgcagctatcgatactggtaatgggaacacatactatcccaccgtagacggccg attcatcatctcccgaggcaacgccaagaactccgtagatctggaaatgaacagcctgacacctgacgacactgccatctactactgtgcggctacgcagggccccctctggcctactttagggactcagttttcgactgaaagttataattactggggccaggggaccctggtcaccgtctcctca.

[0049] The anti-H5 subtype avian influenza virus nanobody protein includes a framework region (FR) and an antibody gene complementarity-determining region (CDR). The framework region is divided into four parts, defined sequentially as FR1, FR2, FR3, and FR4, with amino acid sequences as shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. The complementarity-determining region is divided into three parts, which can be defined sequentially as CDR1, CDR2, and CDR3, with amino acid sequences as shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.

[0050] SEQ ID NO: 3: QVQLQESGGGSVQAGGSLLRLSCSAS;

[0051] SEQ ID NO: 4: WFRQVPGKEREGVAA;

[0052] SEQ ID NO: 5: YYPTVDGRFIISRGNAKNSVDLEMNSLTPDDTAIYYC;

[0053] SEQ ID NO: 6: WGQGTLVTVSS;

[0054] SEQ ID NO: 7: EYTRSMA;

[0055] SEQ ID NO: 8: IDTGNGNT;

[0056] SEQ ID NO: 9: AATQGPLWPTLGTQFSTESYNY.

[0057] Example 3

[0058] Expression of nanobodies

[0059] A full-length Nb10 sequence was synthesized from universal biosynthetic materials, and an 8-His-tag was introduced at the C-terminus. This sequence was cloned into the EcoRI and XbaI sites of the expression vector pPICZαA to construct the recombinant expression plasmid pPICZαA-Nb10. The recombinant plasmid was introduced into *P. pastoris* X33 competent cells using chemical transformation. The transformation product was plated on YPD plates containing 100 μg / ml bleomycin and cultured at 30°C for 3–5 days. Positive clones were screened by PCR using primers 5α-Factor-F and 3AOX1-R. Positive clones were inoculated into BMGY medium and cultured with shaking at 30°C and 250 rpm. OD was monitored. 600 To ensure the logarithmic growth phase, cells were resuspended in BMMY medium and incubated for 3 days at 28-30°C and 250-300 rpm with 1% (v / v) methanol added to induce protein expression. Methanol was added every 24 hours to a final concentration of 0.5%. After three days, the supernatant was collected by centrifugation at 5000g for 10 minutes at 4°C and purified using a Ni-NTA affinity chromatography column. The supernatant was then observed. Figure 2 Page diagrams of purified Nb10 nanobody protein show that its molecular weight is approximately 16 kDa. After dialysis to remove imidazole, the protein concentration was determined using the BCA method. Primers are shown in Table 3.

[0060] Table 3

[0061] Primers Sequence (5′-3′) 5α-Factor-F TACTATTGCCAGCATTGCTGC 3AOX1-R GGCAAATGGCATTCTGACAT

[0062] Example 4: Validation of the in vitro bioactivity of broad-spectrum neutralizing nanobodies

[0063] This study used hemagglutination inhibition assay (HI) and microneutralization assays (MN) to verify the bioactivity of broad-spectrum neutralizing nanobodies.

[0064] In the HI assay, purified Nb10 was serially diluted 2-fold in a V-bottom 96-well plate and mixed with 4 HA units of H5 AIV. The plate was incubated at room temperature for 30 minutes, and 1% chicken red blood cells were added to each well. HI-IC 50Defined as the concentration of nanobody that inhibits hemagglutination of 50% of erythrocytes. Evaluation was performed by repeating the measurement three times. Preparation of 4 HA units requires first determining the viral titer, as follows: Dilute 25 μL of allantoic fluid containing the virus in a 96-well V-shaped plate with PBS at a 1:2 ratio. Next, add 25 μL of PBS and 25 μL of chicken erythrocytes diluted to 1% to the virus dilution. Incubate the plate at 4°C for 40 minutes. HA titer refers to the dilution factor of allantoic fluid that completely inhibits hemagglutination of erythrocytes.

[0065] MDCK cells were maintained in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin, and 1% L-glutamine at 37°C and 5% CO2. One day prior to the experiment, 25,000 MDCK cells were added to each well of a 96-well plate. Nb10 was added to each well with a serially double-diluted solution and an equal volume of 100 TCID50. 50 The virus was mixed for 1 hour, then MDCK cells were added at 37°C for 1 hour. The mixture was removed, and the cells were cultured at 37°C for 20 hours in DMEM medium supplemented with 1 μg / ml TPCK-treated trypsin and an appropriate concentration of Nb10. The cells were washed twice with PBS, fixed with 80% ice-cold acetone at -20°C for at least 1 hour, washed three times with PBS, blocked with 3% BSA-PBS for 30 minutes, and then treated with 2% H2O2 for 30 minutes. The cells were then incubated with mouse anti-NP antibody (1:3000) in 3% BSA-PBS at room temperature for 1 hour, followed by 488-conjugated goat anti-mouse IgG (1:5000) in 3% BSA-PBS at room temperature for 1 hour. Visualization was performed using a fluorescence microscope. The signal from uninfected wells represented 100% inhibition, and the signal from infected wells that had not been incubated with the antibody represented 100% infection. All experiments were performed in triplicate. MN-IC 50 This represents a reduction in fluorescence signal to 50% of the level obtained in the control group with 100% viral infection.

[0066] The broad-spectrum neutralizing activity of the nanobody Nb10 was verified using various recombinant H5 viruses, including clades 2.3.2.1 (Re6 / PR8, Re10 / PR8, and Re12 / PR8) and 2.3.4.4 (Re8 / PR8, Re11 / PR8, and Re14 / PR8). All viruses used were inverse recombinant influenza viruses. The rearranged virus was rescued by transfecting a plasmid containing HA and the remaining seven influenza virus genes derived from A / Puerto Rico / 8 / 34 (H1N1) into co-cultured 293T and MDCK cells using Lipofectamine 2000. 72 hours after transfection, the culture medium was inoculated into chicken embryos or MDCK cells. The original virus multiplied in the allantoic cavity of 10-day-old embryonic eggs. The allantoic fluid containing the virus was harvested, aliquoted, and stored at -80°C. Viral content was determined by a standard hemagglutination assay. The HA gene from the second passage of the rearranged virus was sequenced to confirm the introduced HA gene. Re6 / PR8, Re8 / PR8, Re10 / PR8, Re11 / PR8, Re12 / PR8, and Re14 / PR8 were constructed, and their HA genes were derived from viruses such as A / duck / Guangdong / S1322 / 2010, A / chicken / Guizhou / 4 / 2013, A / duck / Anhui / S1246 / 2014, A / duck / Guizhou / S4184 / 2017, A / chicken / Liaoning / SD007 / 2017, and A / whooperswan / Shanxi / 4-1 / 2020.

[0067] Results of hemagglutination inhibition titer and neutralizing concentration test of nanobody Nb10 against various recombinant viruses

[0068] The specific test results are shown in Table 4-5:

[0069] Table 4

[0070]

[0071]

[0072] Table 5

[0073]

[0074] Re6 / PR8, Re10 / PR8, and Re12 / PR8 viruses belong to branch 2.3.2.1, while Re8 / PR8, Re11 / PR8, and Re14 / PR8 viruses belong to branch 2.3.4.4. Overall, Nb10 exhibits slightly better neutralizing activity against H5AIV of branch 2.3.4.4 than branch 2.3.2.1, and demonstrates the best hemagglutination inhibition and neutralizing activity against Re14 / PR8 virus, achieving HI-IC. 50 and MN-IC 50 The values ​​were 0.05 and 0.01 μg / ml, respectively;

[0075] Meanwhile, to our surprise, during the yeast two-hybrid screening process, we used the guide H1 sequence of the Re8 virus as bait to screen for antibodies. At the same time, a characteristic of the yeast two-hybrid system is that the antibodies obtained from screening have extremely high specificity compared to the bait.

[0076] Based on this, when we consider HI-IC 50 MN-IC 50 During the testing process, it was found that although it had extremely excellent hemagglutination inhibition titer and neutralization concentration against Re8 / PR8 virus, its hemagglutination inhibition titer and neutralization concentration were even better than those against Re14 / PR8 virus. At the same time, its hemagglutination inhibition titer and neutralization concentration against Re11 virus, which is in the same branch as Re8 and Re14, were far inferior to those against the two. Its hemagglutination inhibition titer and neutralization concentration against Re11 virus were almost 20 times that against Re8 virus.

[0077] Its hemagglutination inhibition titer and neutralizing concentration against Re14 virus are almost 50% of those against Re8 virus;

[0078] This result exceeded our expectations because, on the one hand, we screened antibodies using the HA1 sequence of the Re8 virus, and on the other hand, the yeast two-hybrid system exhibits extremely high specificity between antibodies and decoys. Theoretically, we should have obtained an antibody specifically suited for the Re8 virus. However, this antibody showed a more significant neutralizing ability against Re14. It is speculated that this phenomenon may be due to the fact that the nanobody recognition site is not hindered by glycosylation. Nanobodies identified using the yeast two-hybrid system may interact with Re8-HA1 without glycosylation, but the HA1 protein contains multiple glycosylation sites. The RBS region of Re8-HA1 recognized by Nb10 has an N-glycosylation modification site, while the RBS region of Re14-HA1 lacks this feature; thus, Nb10's neutralizing effect on Re14 / PR8 is stronger than that on Re8 / PR8.

[0079] Example 5: In vivo bioactivity verification of broad-spectrum neutralizing nanobodies

[0080] To further evaluate the protective potential of Nb10 against mice infected with H5 subtype AIV, we administered 10 ml of D... 50 Six- to eight-week-old female mice (n=5) were infected with Re6 / PR8, Re8 / PR8, Re10 / PR8, Re11 / PR8, and Re14 / PR8 viruses. Twenty-four hours post-infection, the Re8 / PR8 and Re14 / PR8 groups received Nb10 via intratracheal nebulization at doses of 2.5 mg / kg or 3.5 mg / kg, respectively, while the Re6 / PR8, Re10 / PR8, and Re11 / PR8 groups received Nb10 via intratracheal administration at doses of 4 mg / kg or 8 mg / kg. Animals that lost more than 25% of their initial body weight were immediately euthanized by CO2 asphyxiation and recorded as non-survivors. The control group received PBS (placebo) via intratracheal nebulization. Mortality and changes in body weight over up to 14 days post-infection were observed daily, referencing [reference data]. Figure 3-12 The protective effect of Nb10 is dose-dependent: 2.5 mg / kg Nb10 protected 80% of Re8 / PR8-infected mice from death and prevented significant weight loss in mice under lethal attack; 3.5 mg / kg Nb10 provided 100% protection and significant weight gain. Figure 5-6 Nb10 at doses of 2.5 mg / kg or 3.5 mg / kg provided complete protection in mice infected with Re14 / PR8, and the mice began to show an increasing weight trend on the fourth day post-infection. Figure 11-12 In in vivo protective experiments, Nb10 showed the best neutralizing protective effect against Re14 / PR8. 4 mg / kg of Nb10 provided 60% protection in mice infected with Re10 / PR8, while 8 mg / kg achieved 80% protection. However, the mice exhibited significant weight fluctuations. Figure 7-8 At 4 mg / kg, mice infected with Re6 / PR8 and Re11 / PR8 received only 20% and 40% protection, respectively, but at 8 mg / kg, the protection rates reached 60% and 80%, respectively. The mice also showed minimal weight gain in the later stages of the experiment. Figure 3-4 (and 9-10). All PBS-treated control animals exhibited sustained weight loss after viral challenge and died within 8 days of infection. These results indicate that passive immunization with Nb10 provides mice with good neutralizing protection against avian influenza viruses of subtypes 2.3.2.1 and 2.3.4.4 of the H5 subtype.

Claims

1. A nanobody against H5 subtype avian influenza virus, characterized in that, The amino acid sequence is shown in SEQ ID NO:

1.

2. The use of the anti-H5 subtype avian influenza virus nanobody as described in claim 1 in the preparation of a drug against H5 subtype avian influenza virus.

3. The use according to claim 2, characterized in that, The anti-H5 subtype avian influenza virus drug specifically refers to drugs targeting branches 2.3.2.1 and / or 2.3.4.4 of the H5 subtype avian influenza virus.

4. The use according to claim 2, characterized in that, The drug used to treat H5 subtype avian influenza virus is specifically a drug used to treat H5-Re14 virus.

5. A drug for treating H5 subtype avian influenza virus, characterized in that, It contains the anti-H5 subtype avian influenza virus nanobody as described in claim 1.

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