A polypeptide having broad-spectrum antiviral activity and applications thereof

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to a polypeptide with broad-spectrum antiviral activity and application. The amino acid sequence of the polypeptide comprises at least one of SEQ ID NO: 1-10 or a derivative obtained from the same. The polypeptide acting on LV uses a concentration of 20 μM; the polypeptide acting on H1N1 uses a concentration of 50 μM. The innovative mechanism is to directly act on the virus particles, interfere with the packaging and assembly process, and cause the virus to form non-infective defective particles. In vitro experiments prove that polypeptides such as KV7 and DK8 can dose-dependently destroy the virus morphology, and the inhibition rate of polypeptides such as KD5, VV14 and KV14 on H1N1 is more than 95%. Animal experiments prove that after the polypeptide KD5 is premixed with the virus, the polypeptide is administered by inhalation, and the virus replication in the lung tissue of the infected mice can be significantly inhibited. The present application has the outstanding advantages of broad-spectrum high efficiency, novel mechanism, and suitability for inhalation administration.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a polypeptide with broad-spectrum antiviral activity and its applications. Background Technology

[0002] Influenza A viruses (IAV) are enveloped, single-stranded, negative-sense RNA viruses (PMID: 36680158), classified based on antigenic differences in their nucleoprotein (NP) and matrix protein 1 (M1) (PMID: 36016306). This virus can infect multiple hosts and cause large-scale epidemics through cross-species transmission (PMID: 30487536). Its subtypes are determined by antigenic variations in hemagglutinin (HA) and neuraminidase (NA). Currently, 18 HA subtypes (H1-H18) and 11 NA subtypes (N1-N11) have been identified (PMID: 34326849), among which the H1N1 subtype (such as A / H1N1pdm09) is a key pathogen in several influenza pandemics.

[0003] IAV enters the human body through the respiratory tract, infecting host epithelial cells (PMID: 31714898, PMID: 37112812). Its life cycle includes endocytosis into the cell, membrane fusion triggered by low pH (approximately 6.0) in the endosome (PMID: 12883000), followed by conformational changes in the M1 protein that promote viral core release and uncoating (PMID: 25165113). Viral replication can induce massive cytokine production, triggering a "cytokine storm" and leading to an excessive immune response. Clinical manifestations include fever, cough, and respiratory distress, which can progress to pneumonia, acute respiratory distress syndrome, multiple organ failure, and even death in severe cases (PMID: 32668454, PMID: 33782861, PMID: 37758692). Furthermore, infection with pandemic H1N1 strains is also associated with an increased risk of developing narcolepsy type 1 (PMID: 37188663, PMID: 36311717).

[0004] IAV is one of the leading causes of viral pneumonia and related deaths worldwide (PMID: 21088086, PMID: 31714898). Current treatment focuses on symptomatic and supportive care, often combined with empirical antibiotics, antiviral drugs, and glucocorticoids (PMID: 32397688). Existing antiviral drugs include neuraminidase (NA) inhibitors and polymerase acid protein (PA) inhibitors, while M2 inhibitors have been withdrawn from clinical trials due to widespread resistance (PMID: 29623652). In addition, several monoclonal antibodies targeting HA are in clinical development (PMID: 28146320). However, the high mutation rate of the virus leads to the emergence of drug-resistant strains, which not only reduces the efficacy of NA and PA inhibitors (PMID: 21483816, PMID: 31768027) but may also weaken the binding affinity of monoclonal antibodies to HA (PMID: 24820965), posing a significant public health challenge (PMID: 32755598).

[0005] For prevention, existing vaccines include inactivated vaccines, live attenuated vaccines, and recombinant subunit vaccines (PMID: 33916924). Inactivated vaccines face challenges such as long production cycles and dealing with viral antigen mutations (PMID: 39197095); although live attenuated vaccines can induce a full immune response, there is a potential risk of virulence reversion (PMID: 31530680, PMID: 37344894).

[0006] Therefore, there is an urgent need to continuously develop broad-spectrum antiviral drugs and new vaccine strategies to address the ongoing evolution and spread of influenza. Summary of the Invention

[0007] The purpose of this invention is to provide a polypeptide with broad-spectrum antiviral activity and its applications. The polypeptide of this invention exhibits strong inhibitory activity against influenza A virus (H1N1) and lentivirus (LV) in vitro, and has broad application prospects in the medical field.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect of the invention, a polypeptide with broad-spectrum antiviral activity is provided, the amino acid sequence of said polypeptide comprising: (a) One of SEQ ID NO: 1-SEQ ID NO: 10; or (b) A polypeptide derived from (a) having one or more amino acid substitutions, deletions, or additions, and possessing broad-spectrum antiviral activity, similar to the polypeptide shown in (a); or (c) is a polypeptide derived from (a) that has at least 85% sequence identity with the polypeptide shown in (a) and has broad-spectrum antiviral activity.

[0009] The specific list of peptides in the above scheme is as follows: Table 1

[0010] Furthermore, the polypeptide includes pharmaceutically acceptable salts thereof, or polypeptides formed by modification of the N-terminus and / or C-terminus.

[0011] Furthermore, the N-terminus and / or C-terminus are modified with one of the following: N-terminal acetylation, C-terminal amidation, linkage to a cell-penetrating peptide, linkage to a fatty acid chain, or linkage to a polyethylene glycol molecule.

[0012] Furthermore, the pharmaceutically acceptable salts include trifluoroacetate, acetate, or hydrochloride.

[0013] Furthermore, the polypeptide includes a pharmaceutically acceptable salt thereof. The pharmaceutically acceptable salt includes, but is not limited to, trifluoroacetate, acetate, or hydrochloride.

[0014] Preferably, the polypeptide is modified with trifluoroacetate or acetate.

[0015] In one preferred embodiment, this invention utilizes a polypeptide drug modified with trifluoroacetate or acetate to prepare a polypeptide complex with broad-spectrum antiviral activity. The resulting polypeptide complex can bind to pathogens such as lentiviruses or influenza A viruses, rendering them infective and exhibiting a significant virus-neutralizing effect, demonstrating the potential for precise viral targeting. While maintaining highly efficient antiviral activity, the polypeptide drug acts on the virus very rapidly, thereby reducing the time cost of viral infection treatment.

[0016] Compared to acetate-modified peptides or other antiviral formulations, its trifluoroacetate-modified form exhibits superior antiviral activity, particularly in inhibiting lentiviruses and influenza A viruses, with minimal cytotoxicity. Furthermore, multiple formulations are available for various routes of administration, including nebulization and nasal drops, providing a safer and more controllable delivery method for antiviral therapy.

[0017] Furthermore, one or more of the amino acid sequences of the polypeptide are D-type amino acids.

[0018] In a second aspect of the invention, a nucleic acid molecule is provided that encodes the aforementioned polypeptide.

[0019] In a third aspect of the invention, a carrier is provided comprising the aforementioned nucleic acid molecule.

[0020] Preferably, the vector is a cloning vector or an expression vector; more preferably, the vector is a plasmid, granule, or bacteriophage.

[0021] In a fourth aspect of the invention, a host cell is provided comprising the said nucleic acid molecule or the said vector.

[0022] Preferably, the host cell is a prokaryotic cell or a eukaryotic cell; more preferably, the prokaryotic cell is an Escherichia coli cell, and the eukaryotic cell is a yeast cell, an insect cell, a plant cell, or an animal cell (such as a mammalian cell, a mouse cell, a human cell, etc.).

[0023] In a fifth aspect of the invention, a pharmaceutical composition is provided comprising a therapeutically effective amount of the said polypeptide and a pharmaceutically acceptable carrier.

[0024] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0025] Furthermore, the excipients include at least one of fillers, disintegrants, binders, lubricants, flavoring agents, and preservatives.

[0026] Furthermore, the dosage form of the drug includes at least one of intravenous injection, inhalation preparation, granules, tablets, pills, capsules, injections, and dispersants.

[0027] Solid dosage forms: granules, tablets, pills, capsules, prepared using fillers (such as lactose), disintegrants (such as sodium carboxymethyl starch), and lubricants (such as magnesium stearate); Liquid dosage forms: injections, dispersants, syrups, using solvents (such as water for injection) and stabilizers (such as mannitol); Gaseous formulations: aerosols, inhalers, containing propellants (such as HFA-134a) and dispersion media; Topical formulations: patches, sprays, using adhesives (such as acrylates) and penetration enhancers (such as azone).

[0028] All of the above dosage forms are achieved through conventional pharmaceutical processes (such as wet granulation and freeze drying) to ensure the stability of the composition.

[0029] Furthermore, the method of administration of the drug is adapted according to the dosage form: Oral administration: Suitable for granules, tablets, pills, capsules, and syrups, 1-2 times daily, at a dose of 10-30 mg / kg; Injection administration: Applicable to injections, including intravenous, intramuscular or subcutaneous injection, with a single dose controlled at 10-30 mg / kg; Inhalation administration: Suitable for aerosols and inhalers, administered via inhalation devices (such as DPIs or Nebulizers), for direct delivery of respiratory tract infections; Topical administration: Suitable for sprays and patches, applied to the nasal or oral mucosa, at a dose of 10-30 mg / kg; Transdermal drug delivery: Suitable for patches, using controlled-release membrane technology to prolong drug release.

[0030] Preferably, the inhalation preparation is a nebulized inhalation solution, a nebulized inhalation suspension, or an inhalation dry powder.

[0031] In a third aspect of the invention, a method for preparing the polypeptide is provided, the method comprising: The crude product of the polypeptide was synthesized using a solid-phase synthesis method; The crude product was purified by reversed-phase high-performance liquid chromatography using a mobile phase system containing trifluoroacetic acid. The elution peak containing the target polypeptide was collected and lyophilized to obtain the polypeptide modified with trifluoroacetic acid.

[0032] In a fourth aspect of the invention, the use of the said polypeptide or the said pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of viral infections is provided.

[0033] Furthermore, the working concentration of the polypeptide is 20 μM-50 μM; Furthermore, for lentivirus LV, the final concentration of the peptide is 20 μM, and for influenza virus H1N1, the final concentration of the peptide is 50 μM.

[0034] Preferably, the working concentration of the peptide provided by the present invention for inhibiting LV_EGFP is not less than 2 mM•l. -1 .

[0035] Preferably, the influenza A virus provided by the present invention is A / Puerto Rico / 8 / 1934_GFP, H1N1_GFP.

[0036] Preferably, the working concentration of the peptide provided by the present invention for inhibiting H1N1_GFP is not less than 5 mM•l. -1 .

[0037] Furthermore, the viral infection includes influenza A virus infection or lentivirus infection.

[0038] Furthermore, the peptides shown in SEQ ID NO:1-SEQ ID NO:10 can all inhibit lentiviral infection. Among them, peptides VV14 (SEQ ID NO:3), DK8 (SEQ ID NO:4), KD5 (SEQ ID NO:5), KV14 (SEQ ID NO:6), KK14 (SEQ ID NO:7), and DV4 (SEQ ID NO:2) can be used to inhibit influenza A virus infection.

[0039] In a sixth aspect of the invention, a method for preventing or treating viral infection in a subject is provided, the method comprising administering a therapeutically effective amount of the polypeptide or the pharmaceutical composition to a subject in need.

[0040] Furthermore, the administration route is nebulized inhalation.

[0041] Furthermore, the concentration of the polypeptide in the premixed formulation with influenza A virus before nebulization is 5 mM, and the premixing time is 5 min.

[0042] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: 1. Broad-spectrum and highly effective antiviral activity: The peptides of this invention exhibit strong inhibitory capabilities against a variety of enveloped viruses. In vitro experiments confirmed that peptides KD5, VV14, and KV14, at a concentration of 50 μM, all showed inhibition rates exceeding 95% against influenza A virus (H1N1_GFP), achieving near-complete neutralization. Simultaneously, at a concentration of 20 μM, peptides KV7, DV4, DK8, KD5, VV14, and KV14 also showed significant inhibitory activity against mammalian gene-expressing lentiviruses (LV_EGFP) (for example, KV7 reduced viral infection efficiency by more than 70%). This indicates that the peptides of this invention possess broad-spectrum antiviral potential against different viral families, providing a new solution to the treatment challenges caused by viral mutations.

[0043] 2. Novel and Unique Mechanism of Action: This invention is the first to reveal the antiviral mechanism of the peptides using transmission electron microscopy (TEM). The study found that peptides KV7 and DK8 can act directly on viral particles in a dose- and time-dependent manner, leading to smaller and fewer viral particles, forming non-infectious defective particles. This indicates that the peptides of this invention do not exert their effects through traditional competitive inhibition of receptors or enzyme activity, but rather by interfering with the assembly or packaging integrity of the viral particles themselves, thereby fundamentally destroying their infectivity. This novel mechanism of action is less likely to induce drug resistance in viruses and has significant clinical value.

[0044] 3. Clear Early Target of Action: Through peptide-virus time-sequential premixing experiments, this invention confirms that the peptide primarily acts on the adsorption / invasion phase of viral infection, rather than the replication phase after the virus enters the cell. This discovery clarifies its window of action and provides a crucial theoretical basis for the timing of clinical drug use (such as post-exposure prophylaxis or early treatment).

[0045] 4. Excellent drug-likeness and stability: This invention employs an optimized reversed-phase high-performance liquid chromatography (RP-HPLC) purification process, obtaining a polypeptide trifluoroacetate with a purity exceeding 95%, exhibiting a fluffy physical form and excellent water solubility. These superior physicochemical properties greatly facilitate the formulation of high-concentration preparations (especially nebulized inhalation formulations), improve the stability and bioavailability of the polypeptide, and lay a solid foundation for its subsequent industrial development and clinical application.

[0046] 5. Innovative Administration Route and Definite In vivo Efficacy: This invention pioneeringly verifies the feasibility and effectiveness of administering the described peptide via nebulized inhalation. Animal experiments show that simply premixing the peptide KD5 with H1N1 virus and then nebulizing it significantly inhibits viral replication in the lung tissue of virus-infected mice (confirmed by IHC and GFP expression detection). This demonstrates that this administration strategy can directly deliver the peptide to the respiratory infection target, with rapid onset and definite efficacy, and is expected to avoid the side effects of systemic administration, providing a direct and efficient new treatment and prevention strategy for respiratory viral infections. Attached Figure Description

[0047] Figure 1 The figure shows the anti-lentiviral activity of the polypeptide of the present invention in HEK-293T cells; (A) The effect of KV7 on the infectivity of LV was detected by flow cytometry. 10 mM KV7 was premixed with 0.5 MOI LV, with a final concentration of 500 μM (KV7:LV premix volume ratio of 12:1) and 250 μM (KV7:LV premix volume ratio of 6:1). LV_EGFP expression was detected by flow cytometry 48 h post-infection. (B) Peptides effective against viral infection were screened using disc confocal microscopy. 2 mM peptide was premixed with 0.5 MOI LV for 30 min, with a final peptide concentration of 20 μM. Scale bar: 20 μm. This indicates that P < 0.05. This indicates that P < 0.001. The P-value was calculated using a two-tailed Student's test.

[0048] Figure 2Physical characterization of lentiviral particles was assessed using zeta potential and transmission electron microscopy. (A) MST time trajectories of 16 capillaries containing the same concentration of LV and increasing concentrations of the visible light-interacting chaperone KV7_FITC were recorded and plotted in the obtained figure (left). Normalized fluorescence of the MST traces was plotted against LV concentration (right). (B) The zeta potential reversal of the surface charge of the peptide-virus plays a crucial role in the antiviral activity of the peptide. Surface potentials were measured using different concentrations of peptide premixed with LV at a peptide:LV premix volume ratio of (10:1). (C) Ctrl indicates ultrapure water premixed with LV at a H2O:LV premix volume ratio of (1:1) without peptide treatment. White arrows indicate small spherical particles. (D) Representative images of LV virus particles treated with high or low concentrations of KV7 were analyzed by TEM. White arrows indicate small spherical particles. (E) Representative images of LV virus particles treated with KV7 for different time periods were analyzed by TEM. White arrows indicate small spherical particles. (F) Representative images of LV virus particles processed with DK8 at different times were analyzed by TEM. White arrows indicate small spherical particles. The right image in each set is a magnified view of a portion of the original image, scale bar, 100 nm.

[0049] Figure 3 Antiviral activity of peptides in Huh7_T cells. (A) Peptides effective against influenza virus were screened by confocal microscopy using a rotating disk. 5 mM peptides were premixed with 0.6 MOI H1N1_GFP for 30 min. Scale bar: 20 μm. This indicates that P < 0.05. P < 0.001. P values ​​were calculated using a two-tailed Student's test. (B) Detection of the temporal effects of VV14 and KV14 on H1N1_GFP. The aforementioned peptides were premixed with 0.6 MOI H1N1_GFP at different time intervals using 5 mM of the aforementioned peptides. Scale bar: 20 μm.

[0050] Figure 4 Results of the detection of a mouse model of influenza virus constructed by nebulization and nasal drops. (A)-(B) IHC of NP in lung tissue at 4 dpi. Scale bar: 50 μm.

[0051] Figure 5 Results of the detection of a mouse model of influenza virus constructed by nebulization and nasal drops. (A)-(B) Ice cleavage of H1N1_GFP in lung tissue at 4 dpi. Scale bar: 100 μm.

[0052] Figure 6Based on the 5-minute premixing condition used in in vitro screening, nebulization of a premixed complex of KD5 / AV7 and H1N1 effectively inhibited viral replication in the lungs of virus-infected mice. (A) Premixing time of KD5 and influenza virus was detected in A549. 5 mM KD5 and 0.6 MOI H1N1_GFP were premixed for different times. (B)-(C) A mouse model of peptide-H1N1 treatment was constructed by nebulization of AV7 and KD5 premixed with H1N1 for 5 min. Ctrl refers to premixing ultrapure water with H1N1, H2O:H1N1 premixed volume ratio (1:1), without peptide treatment. IHC and GFP positive expression of NP in lung tissue at 4 dpi. Scale bar: 50 μm. This indicates that P < 0.05. P < 0.01. The P-value was calculated using a two-tailed Student's t-test. Protein expression statistics: As shown in the figure, there are three treatments, with four replicates per treatment group. For each replicate, six 10× images of different fields of view were randomly selected, resulting in 24 statistical points per treatment group. The %Area was calculated using ImageJ. Detailed Implementation

[0053] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0054] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0055] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0056] The present application will now be described in detail with reference to embodiments and experimental data.

[0057] Example 1: Synthesis and purification of polypeptides This embodiment provides a method for preparing peptides, taking peptides KV7 (SEQ ID NO: 1) and KD5 (SEQ ID NO: 5) as examples. The synthesis methods for other peptides are similar.

[0058] 1. Synthesis: A standard Fmoc solid-phase synthesis strategy was employed on an automated peptide synthesizer. Taking KV7 (sequence Lys-Asp-Asn-Asp-Asp-Ile-Val) as an example, Rink Amide MBHA resin was used as the solid-phase support, and the corresponding Fmoc protected amino acids were sequentially coupled according to the sequence. Each coupling cycle included: deprotection (treatment with 20% piperidine in N,N-dimethylformamide (DMF) solution), washing, and coupling (using Fmoc-amino acids, HBTU / HOBt as activators, and N,N-diisopropylethylamine (DIPEA) as a base). After synthesis, the peptide was cleaved from the resin and the side-chain protecting groups were removed simultaneously using a cleavage mixture (trifluoroacetic acid (TFA):water:phenol:triisopropylsilane = 92:2:3:3, v / v) with stirring at room temperature for 2-3 hours.

[0059] 2. Purification: The crude polypeptide was precipitated with cold methyl tert-butyl ether and collected by centrifugation to obtain the crude polypeptide. The crude product was dissolved in water containing 0.1% TFA and purified using a preparative reversed-phase high-performance liquid chromatography (RP-HPLC) system. The chromatographic conditions were as follows: Column: C18 column (e.g., Waters XBridge Prep C18, 5 μm, 19 × 150 mm); Mobile phase A: aqueous solution containing 0.1% TFA, Mobile phase B: acetonitrile solution containing 0.1% TFA; Gradient elution: Mobile phase B was linearly increased from 20% to 50% within 30 minutes; Detection wavelength: 220 nm. The main chromatographic peak was collected.

[0060] 3. Freeze-drying: The collected pure solution was freeze-dried under vacuum to obtain a white, fluffy, amorphous powder of polypeptide trifluoroacetate. Analytical HPLC analysis showed that its purity was greater than 95%.

[0061] Using the same method, peptides such as DV4 (SEQ ID NO: 2), VV14 (SEQ ID NO: 3), DK8 (SEQ ID NO: 4), KV14 (SEQ ID NO: 6), and KK14 (SEQ ID NO: 7) were successfully synthesized, and their purity all met the requirements.

[0062] In specific embodiments of the present invention, for ease of illustration, the polypeptide KV7-1 shown in SEQ ID NO:1 is illustrated in acetate form, and the polypeptides shown in SEQ ID NO:2-SEQ ID NO:10 are illustrated in trifluoroacetate form. Those skilled in the art will understand that other pharmaceutically acceptable salt forms are also applicable to the present invention.

[0063] Example 2: In vitro antiviral activity assay (for lentivirus LV_EGFP) This embodiment verifies the inhibitory effect of the peptide of the present invention on lentiviruses.

[0064] 1. Cells and Viruses: The human embryonic kidney HEK-293T cell line was used. The virus was a mammalian gene-expressing lentiviral particle (LV_EGFP) carrying the enhanced green fluorescent protein (EGFP) reporter gene.

[0065] 2. Experimental Procedure: HEK-293T cells in good growth condition were seeded into 96-well plates. The test peptides (KV7, DV4, VV14, DK8, KD5, KV14, etc.) were premixed with the virus, and then diluted with serum-free medium to different working concentrations (including 20 μM, 500 μM, 250 μM, etc.). The peptide solutions were mixed with LV_EGFP virus solution (MOI = 0.5) at a 1:1 volume ratio and pre-incubated at room temperature for 30 minutes. This mixture was then added to the cells for further culture. Cells infected with the virus without peptides were set up as positive controls (100% infection), and uninfected cells were set up as negative controls.

[0066] 3. Detection and Results: After 48 hours of infection and culture, cells were collected by trypsin digestion, and the percentage of EGFP-positive cells was detected by flow cytometry. The relative infection inhibition rate of each peptide treatment group was calculated with the infection rate of the positive control being 100%.

[0067] The results are as follows Figure 1 As shown, compared with the positive control, all tested peptides (KV7, KV7-1, DV4, VV14, DK8, KD5, KV14, KK14, AV7, CC9, GG7) exhibited significant anti-lentiviral activity, with the KV7 peptide treatment group showing a reduction in viral infection efficiency of over 70%. This indicates that the peptides of the present invention can effectively inhibit lentiviral infection.

[0068] Example 3: Study on the anti-lentiviral mechanism of peptides KV7 and DK8 - based on zeta potential and transmission electron microscopy analysis 1. Objective: This embodiment aims to investigate the mechanism of action of the peptides KV7 and DK8 against lentiviruses (LV) from a biophysical and morphological perspective. By measuring changes in viral particle surface potential, peptide-virus binding affinity, and alterations in viral morphology, its unique mode of action is elucidated.

[0069] 2. Experimental Materials and Methods (1) Virus and peptide: mammalian gene-expressing lentiviral particles (LV); peptide KV7 (sequence shown in SEQ ID NO:1) and its fluorescent label KV7_FITC; peptide DK8 (sequence shown in SEQ ID NO:4).

[0070] (2) Main instruments: Micro thermophoresis apparatus (MST), Zeta potential analyzer, transmission electron microscope (TEM).

[0071] (3) Experimental methods: ① Micro-thermophoretic (MST) binding assay: LV virus particles at a fixed concentration were mixed with KV7_FITC fluorescently labeled peptides at increasingly higher concentrations. Fluorescence changes over time were measured and recorded using an MST instrument in 16 capillaries. The binding dissociation constant (Kd) was calculated by analyzing the relationship between the normalized fluorescence value and the peptide concentration.

[0072] ②Zeta potential measurement: LV virus particles were premixed with different concentrations of peptide KV7 at a volume ratio of 10:1, incubated at room temperature, and the surface potential (Zeta potential) change of the mixture was directly measured using a Zeta potential analyzer.

[0073] ③ Transmission Electron Microscopy (TEM) Sample Preparation and Observation: Control group: Ultrapure water and LV virus stock solution were mixed at a volume ratio of 1:1 and incubated at room temperature for 30 minutes.

[0074] Processing Group: (a) Dose-dependent: LV virus stock solution was mixed with low and high concentrations of KV7 peptide solution in proportion.

[0075] (b) Time-dependent: LV virus stock solution was mixed with KV7 or DK8 peptide solution and incubated for different times (e.g., 5, 15, 30 minutes).

[0076] The sample treated as described above was dropped onto a copper grid of a carbon support film, and after being negatively stained with 2% phosphotungstic acid, it was observed and images were captured under TEM.

[0077] 3. Experimental Results The results are as follows Figure 2 As shown.

[0078] Figure 2 MST binding curve (A): The MST time trajectory and fitting curve show that KV7_FITC binds to LV virus particles in a concentration-dependent manner. The calculated binding dissociation constant (Kd) is (5.14 ± 4.98) μM, which proves that KV7 and LV virus particles have a high-affinity direct interaction.

[0079] Figure 2 Zeta potential analysis (B): Compared with untreated virus (control group), the surface zeta potential of LV virus particles treated with peptide KV7 was significantly reduced (shifted towards negative values), and this effect was concentration-dependent. This indicates that peptide KV7 disrupts the electrostatic stability of virus particles by neutralizing the positive charge on their surface.

[0080] Figure 2 CF (TEM morphological observation): Figure 2 In the control group (C), the LV virus particles treated with ultrapure water were morphologically intact, uniform in size (approximately 20-60 nm), and had a clear structure.

[0081] Figure 2 Medium D (dose-effect): After treatment with low concentrations of KV7, the morphology of viral particles changed significantly, with a large number of smaller (approximately 20-30 nm) spherical particles appearing. High concentrations of KV7 treatment led to a sharp reduction in the number of viral particles, almost completely disintegrating them into even smaller fragments.

[0082] Figure 2 E (KV7 time effect): The destructive effect of KV7 on the viral structure is time-dependent; as the processing time increases, the degree of viral particle fragmentation intensifies.

[0083] Figure 2 DK8 time effect: The peptide DK8 can also disrupt the viral particle structure in a time-dependent manner, and can induce the production of a large number of fine particles in a shorter time. Its action kinetics are faster than those of KV7.

[0084] In conclusion: This embodiment reveals the antiviral mechanism of the peptides KV7 and DK8 of the present invention through comprehensive biophysical and morphological analysis: Direct binding: The peptide KV7 can bind directly to LV virus particles with high affinity (Kd = 5.14 ± 4.98 μM).

[0085] Neutralizing charge: After binding, the peptide can effectively neutralize the positive charge on the surface of the virus particle, disrupting its electrostatic balance.

[0086] Structural disruption: Ultimately, the structural integrity of the viral particles is disrupted in a dose- and time-dependent manner, dissociating into non-infectious defective particles. Transmission electron microscopy (TEM) results show that peptides KV7 and DK8 can directly act on mature viral particles, disrupting the integrity of the viral envelope and / or core structure, causing them to lyse into non-infectious defective particles, thereby achieving viral inactivation. This mechanism of action differs from traditional receptor competition or enzyme inhibition modes, representing a novel, physical viral inactivation method, providing crucial evidence for the inventiveness and novelty of this invention.

[0087] Example 4: Evaluation of the in vitro anti-influenza A virus (H1N1) activity and timing effect of the peptide 1. Objective: This embodiment aims to quantitatively evaluate the inhibitory effect of the series of peptides described in this invention on influenza A virus (H1N1_GFP) infection using a human Huh7_TMPRSS2 (Huh7_T) cell model, and further explore the optimal pre-incubation time for the effective peptides to exert their inhibitory effect, providing key basis for elucidating their mechanism of action and subsequent in vivo experimental design. 2. Experimental Materials and Methods (1) Cells and viruses: Human hepatocellular carcinoma Huh7 cells (Huh7_T) overexpressing TMPRSS2; recombinant influenza A virus A / Puerto Rico / 8 / 1934 (H1N1_GFP) carrying green fluorescent protein (GFP) reporter gene.

[0088] (2) Polypeptides: The polypeptides CC9, AV7, GG7, KV7_1, KV7, KD5_1, DV4, KK14, DK8, KD5, VV14, and KV14 described in this invention (for the sequence details, please refer to the sequence list in the specification).

[0089] (3) Experimental steps: ① Antiviral activity screening: Healthy Huh7_T cells were seeded into confocal culture plates. Each 5 mM peptide to be tested was mixed with H1N1_GFP virus solution (MOI = 0.6) at a 1:1 volume ratio, diluted to a working concentration of 50 μM with cell maintenance medium, and pre-incubated at room temperature for 30 minutes. The original culture medium in the cell culture plate was discarded, and 100 μL of the above peptide-virus mixture was added to each well to infect the cells. After an appropriate incubation period, the cells were tested.

[0090] ② Time-series effect detection: The effective peptides VV14 and KV14 (5 mM) selected above were mixed with H1N1_GFP virus solution (MOI = 0.6) and pre-incubated at room temperature for different times (time gradient according to...). Figure 3 (B shows the settings), and the subsequent infection steps are the same as above.

[0091] ③ Detection and analysis methods: Live cells were scanned and imaged using a rotating confocal microscope to observe and collect GFP fluorescence signals from cells in each well.

[0092] Image analysis software was used to quantitatively analyze the signal intensity of fluorescence images, and the virus positivity rate or relative fluorescence intensity was calculated. Experimental data are expressed as mean ± standard deviation. Statistical analysis was performed using a two-tailed Student's t-test, with p < 0.05 and p < 0.001 considered statistically significant.

[0093] 3. Experimental Results The results are as follows Figure 3 As shown.

[0094] Figure 3 Medium A (antiviral activity screening): Fluorescence microscopy images: Compared with the virus control group (H2O), the GFP fluorescence signal intensity of cells treated with different peptides (such as KD5, VV14, KV14, etc.) was significantly reduced.

[0095] Quantitative statistical bar chart: Quantitative analysis of fluorescence intensity showed that the virus positivity rate / fluorescence intensity in the treatment groups of peptides KD5, VV14, and KV14 was significantly lower than that in the virus control group (p<0.001). Peptides DK8 and KK14 also showed significant inhibitory effects (p<0.05). However, there was no statistically significant difference between the treatment groups of peptides CC9 and AV7 and the virus control group.

[0096] The results demonstrate that multiple peptides, such as KD5, VV14, and KV14, in the peptides of this invention can effectively inhibit the infection of host cells by H1N1 virus in vitro.

[0097] Figure 3 B (Time-sequential effect detection): Fluorescence microscopy images showed that the inhibitory effects of peptides VV14 and KV14 after different pre-incubation times with the virus varied with the pre-incubation time.

[0098] The results showed that VV14 and KV14 could be incubated for a relatively short period of time (the specific time depended on...). Figure 3 B-mode (as shown in the image) can exhibit a significant inhibitory effect, and its antiviral activity is time-dependent.

[0099] In conclusion: This embodiment demonstrates, using rotary confocal imaging, that the peptides KD5, VV14, and KV14 of this invention can significantly inhibit influenza A virus (H1N1) infection, with KD5, VV14, and KV14 showing particularly outstanding inhibitory effects. Furthermore, the antiviral effects of the effective peptides VV14 and KV14 are time-dependent, suggesting a possible direct interaction with viral particles. These results provide important data support for screening lead peptides and determining their optimal in vitro action conditions.

[0100] Example 5: Establishment of a mouse model of influenza virus and evaluation of the dose-response relationship (IHC detection NP) 1. Objective: This embodiment aims to establish a reproducible mouse nebulization model of influenza A virus (H1N1) infection by comparing nebulized inhalation and traditional nasal inoculation, and to systematically evaluate the effects of different viral inoculation doses on viral replication levels in lung tissue (indicated by nucleoprotein NP expression), thereby determining the optimal infection conditions suitable for subsequent in vivo pharmacodynamic studies of antiviral drugs.

[0101] 2. Experimental Materials and Methods Experimental animals: 6-8 week old SPF grade BALB / c female mice, randomly grouped.

[0102] Virus strain: Influenza A virus A / Puerto Rico / 8 / 1934 (H1N1_PR8) strain, titer determined after amplification in chicken embryos.

[0103] Virus inoculation: Mice were randomly divided into a negative control group (Ctrl, inoculated with sterile PBS buffer) and an experimental group. The experimental group was inoculated with different doses of virus solution (50 μL) via intranasal drop, with dose gradients including: 5, 40, 200, 1000, and 1 × 10⁻⁶. 4 5×10 4 1×10 5 5×10 5 PFU / 50μL. Simultaneously, supplemental exposure to viral aerosols via nebulized inhalation was administered to simulate natural respiratory infection.

[0104] Mice were lightly anesthetized before inoculation to ensure a smooth procedure.

[0105] Sample collection and processing: Mice were euthanized on day 4 post-infection (4 dpi), and lung tissue was completely removed. The lung tissue was fixed in 4% paraformaldehyde solution for 24 hours, followed by paraffin embedding. The paraffin blocks were serially sectioned (4 μm thick) and mounted on glass slides for later use.

[0106] Immunohistochemical (IHC) staining: After dewaxing and hydration, sections underwent antigen retrieval. Incubation was performed overnight with a mouse monoclonal primary antibody against influenza virus nucleoprotein (NP). The reaction was then performed using HRP-labeled secondary antibody and a DAB staining kit, with hematoxylin counterstaining of cell nuclei. After mounting with neutral resin, the sections were observed and images acquired under a light microscope.

[0107] Image analysis: Five non-overlapping fields of view were randomly selected from each group, and the percentage area (%Area) of NP-positive stained regions was analyzed using ImageJ software. Data are expressed as mean ± standard deviation, and one-way ANOVA was used for inter-group comparisons. p < 0.05 is considered statistically significant.

[0108] 3. Experimental Results The results are as follows Figure 4 As shown.

[0109] Figure 4 China A and Figure 4 Image B (IHC staining): Negative control group (Ctrl): The lung tissue of mice was structurally intact, with only background staining and no obvious NP protein-specific brown staining.

[0110] Virus inoculation group: NP positive signals of varying degrees were observed in the lung tissue of mice in all virus-inoculated experimental groups. As the inoculation dose increased from 5 PFU / 50 μL to 1000 PFU / 50 μL, the intensity and extent of NP positive staining showed a significant dose-dependent increase.

[0111] Starting at a dose of ≥1000 PFU / 50μL, a large, dense area of ​​strong positive staining was observed, indicating that the virus was replicating extensively in the lungs.

[0112] Quantitative analysis: The statistical results of IHC-positive area were consistent with morphological observations, and the expression level of viral NP protein was significantly positively correlated with the inoculation dose. p<0.05).

[0113] In summary, this embodiment successfully constructed a mouse model of H1N1 influenza A virus infection through a combination of nebulization and nasal drops, and clarified the quantitative relationship between the viral dose and the viral load in the lungs. The results show that this model can produce a clear and highly reproducible infection phenotype, providing a crucial and reliable experimental platform for evaluating the in vivo efficacy of antiviral peptides in subsequent embodiments. Based on this dose-response relationship, a viral dose (≥1000 PFU / 50 μL / 50 μL) inducing moderate infection was subsequently selected for pharmacodynamic evaluation to accurately observe the intervention effect of the therapeutic drugs.

[0114] Example 6: Assessment of Influenza Virus Infection Level in a Mouse Model Based on the GFP Reporter Gene (GFP Fluorescence Detection) 1. Purpose of the invention: This embodiment aims to visually display and quantitatively evaluate the viral replication and distribution in the lung tissue of mice after different doses of influenza A virus (H1N1_GFP) were administered via frozen section and fluorescence imaging technology, comparing nebulization and traditional drop methods, and subsequently inoculating mice with nebulization. This verifies the reliability of the constructed animal model and the viral dose-response relationship, providing a visual indicator for monitoring viral load in subsequent in vivo pharmacodynamic studies.

[0115] 2. Experimental Materials and Methods Experimental animals: 6-8 week old SPF grade BALB / c female mice, randomly grouped.

[0116] Virus strain: Recombinant influenza A virus A / Puerto Rico / 8 / 1934 (H1N1_GFP) carrying the green fluorescent protein (GFP) reporter gene.

[0117] Virus inoculation: Mice were randomly divided into a negative control group (inoculated with sterile PBS buffer) and an experimental group. The experimental group was inoculated with different doses (50 μL) of H1N1_GFP virus solution via intranasal drop, with dose gradients including: 5, 40, 200, 1000, and 1×10⁻⁶. 4 5×10 4 1×10 5 5×10 5 PFU / 50μL (based on) Figure 5 (Dosage settings shown). Simultaneously, mice were exposed to viral aerosols via nebulized inhalation to simulate natural respiratory infection. Prior to inoculation, mice were lightly anesthetized (e.g., by isoflurane inhalation) to ensure smooth operation.

[0118] Sample collection and processing: Mice were euthanized on day 4 post-infection (4 dpi), and lung tissue was completely removed. The lung tissue was immediately placed in OCT embedding medium, rapidly frozen in liquid nitrogen, and stored at -80°C. Serial sections (10 μm thickness) were prepared using a cryostat, mounted on pre-cooled glass slides, and stored at -20°C for later use.

[0119] Fluorescence Imaging and Analysis: After slides equilibrate to room temperature, cell nuclei are counterstained with DAPI (1 μg / mL) for 5 minutes, washed with PBS, and mounted. Images are observed and acquired using a rotating confocal microscope or fluorescence microscope under the GFP channel (excitation 488 nm, emission 500-550 nm). Microscope parameters are kept consistent to ensure comparability. Five non-overlapping fields of view are randomly selected from each group, and the integrated fluorescence intensity (Integrated Density) or percentage of positive area (%Area) of the GFP fluorescence signal is quantitatively analyzed using ImageJ software.

[0120] Statistical analysis: Data are expressed as mean ± standard deviation. One-way ANOVA was used for inter-group comparisons. p < 0.05 is considered statistically significant.

[0121] 3. Experimental Results The results are as follows Figure 5 China A and Figure 5 As shown in B: Negative control group (PBS): The lung tissue of mice was structurally intact, with only weak background fluorescence and no specific GFP signal, indicating no viral replication.

[0122] Virus inoculation group: Green fluorescence signals of varying degrees were observed in the lung tissue of all mice in the experimental group inoculated with H1N1_GFP virus. As the dose of c increased from ≥1000 PFU / 50μL, the intensity, brightness, and distribution range of the GFP fluorescence signal showed a significant dose-dependent increase.

[0123] At a dose of 1000 PFU, the fluorescence signal intensity increased, and GFP fluorescence was observed.

[0124] In the high-dose group (e.g., 1×10⁻⁶) 4 -5×10 5 Within the PFU dose range, a large area of ​​dense, bright green fluorescence is visible, covering most of the lung lobes, indicating extensive viral replication and spread within the lungs. A high-magnification image (scale bar 100 μm) clearly shows the focal point of viral replication at the cellular level.

[0125] Quantitative analysis: The statistical results of GFP fluorescence intensity were consistent with morphological observations, and the viral replication level (expressed as fluorescence intensity) was significantly positively correlated with the inoculation dose. p<0.05). There was no significant difference between the low-dose group and the negative control group, while there were statistically significant differences between the medium- and high-dose groups and the low-dose group and the control group.

[0126] In summary, this embodiment, through fluorescence imaging of the H1N1_GFP reporter gene, directly and quantitatively confirmed a significant dose-response relationship between the replication degree of influenza A virus in mouse lung tissue and the inoculation dose. This result not only validates the high reliability and gradient of the mouse infection model constructed through combined nebulization and intranasal inoculation, but also provides a sensitive and reliable visual monitoring indicator of viral load for subsequent in vivo efficacy evaluation of antiviral peptides (e.g., by comparing the degree of fluorescence attenuation between the treatment group and the model group). Based on this model, a viral dose ≥1000 PFU / 50 μL inducing moderate infection will be selected for subsequent pharmacodynamic experiments to facilitate accurate assessment of the drug's therapeutic effect.

[0127] Example 7: Optimization of premixing time between peptide KD5 and virus and verification of in vivo antiviral efficacy 1. This embodiment aims to screen the shortest effective pre-incubation time of peptide KD5 with influenza A virus (H1N1) using an in vitro cell model. Based on optimized conditions, an in vivo animal model was further used, with the control peptide AV7 (which has no antiviral activity) as a negative control, to systematically evaluate the inhibitory effect of peptide KD5 on virus-infected lung tissue after nebulized inhalation, providing experimental evidence for the development of clinical dosing regimens.

[0128] 2. Experimental Materials and Methods Cells, Viruses, and Animals: Human lung cancer A549 cell line; recombinant influenza A virus A / Puerto Rico / 8 / 1934 (H1N1_GFP) carrying the green fluorescent protein (GFP) reporter gene.

[0129] 6-8 week old SPF grade BALB / c female mice were randomly assigned to groups.

[0130] Polypeptides: The present invention comprises the polypeptides KD5 (sequence as shown in SEQ ID NO: 5) and AV7 (sequence as shown in SEQ ID NO: 2).

[0131] 3. Optimization experiment of in vitro premixing time: (1) Test methods A549 cells in good growth condition were seeded into 96-well plates.

[0132] The KD5 peptide was diluted to a working concentration of 5 mM with serum-free medium and mixed with an equal volume of H1N1_GFP virus solution (MOI = 0.6). The mixture was then pre-incubated at room temperature for 5, 10, 20 and 30 minutes, respectively.

[0133] The above premixed complex was added to cells for infection, and after culturing for an appropriate time, GFP fluorescence signal images were observed and acquired using a fluorescence microscope.

[0134] Using the virus infection group without added peptides as a positive control (100%), the relative inhibition rate at each time point was calculated.

[0135] (2) Experimental results Figure 6 In the A549 cell model, premixing KD5 peptide (5 mM) with H1N1_GFP virus for 5 minutes showed a significant inhibitory effect on the virus. Extending the premixing time to 30 minutes did not further enhance the inhibitory effect, indicating that a short premixing time (5 minutes) is sufficient for KD5 to fully exert its antiviral activity.

[0136] 4. In vivo pharmacodynamic experiments: (1) Approximately 18-20 g BALB / c female mice were randomly divided into a control group (Ctrl) and a polypeptide treatment group, with 4 mice in each group: Experimental Group 1: 5 mM peptide AV7 and H1N1 influenza virus suspension were premixed and incubated in vitro for 5 minutes, and then administered to mice via nebulizer to infect them.

[0137] Experimental Group 2: 5 mM peptide KD5 and H1N1 influenza virus suspension were premixed and incubated in vitro for 5 minutes, and then administered to mice via nebulizer to infect them.

[0138] The control group (Ctrl) was inoculated with an equal volume of ultrapure water and H1N1 influenza virus premixed (volume ratio 1:1) via nebulization, without the addition of peptides.

[0139] Lung tissue was taken on day 4 post-infection (4 dpi) for pathological analysis, including immunohistochemical (IHC) staining of viral nucleoprotein (NP) and detection of green fluorescent protein (GFP) carried by H1N1, to assess viral infection status.

[0140] (2) Nebulized drug administration and infection: The above premixed compound was nebulized using a vibrating sieve nebulizer, and the mice were inhaled into the aerosol in a closed system to complete the infection.

[0141] (3) Sample collection and testing: Mice were sacrificed on the 4th day after infection (4 dpi) and lung tissue was collected.

[0142] (4) Detection indicators: Mice were euthanized on the 4th day after infection (4 dpi) and lung tissue was collected.

[0143] Immunohistochemistry (IHC): Partial lung tissue was fixed, embedded in paraffin, sectioned, and stained with a specific antibody against influenza virus nucleoprotein (NP) to observe the distribution and load of the virus in the lung tissue.

[0144] Fluorescence imaging: The fluorescence signal of H1N1_GFP in some fresh lung tissue was directly observed through a fluorescence imaging system to assess viral replication.

[0145] Image statistical analysis: ImageJ software was used to quantitatively analyze the positive staining area (%Area) of IHC-stained sections. Multiple fields of view were randomly selected for each group for statistical analysis. ImageJ software was used to calculate the percentage of NP-positive areas (%Area) in IHC staining.

[0146] All data are expressed as mean ± standard deviation. Two-tailed Student's t-tests were used for intergroup comparisons, with p < 0.05 considered acceptable. p < 0.01 is considered statistically significant.

[0147] (5) Experimental results IHC staining results ( Figure 6 (B) In the control group (Ctrl), extensive and intense brownish-red NP protein staining was observed in the lung tissue of mice, indicating extensive viral replication. In contrast, the NP-positive staining area in the lung tissue of mice treated with AV7 and KD5 was significantly reduced, with the reduction being particularly pronounced in the KD5 group.

[0148] GFP fluorescence results ( Figure 6 (C) The lung tissue of the control group showed a strong green fluorescent signal, while the fluorescence signal intensity of the AV7 and KD5 treatment groups was significantly reduced.

[0149] Quantitative statistical analysis: Quantitative calculation of the IHC-positive area showed that, compared with the control group, the viral NP expression in the AV7 treatment group was significantly reduced (p<0.05), while the reduction in the KD5 treatment group was even greater, with a highly significant difference. p<0.01). The quantitative analysis results of GFP fluorescence intensity were consistent with those of IHC.

[0150] In summary, this embodiment determined through in vitro experiments that the minimum effective premixing time required for the antiviral effect of peptide KD5 is 5 minutes. In vivo experiments further confirmed that, based on this optimized condition, administration of the premixed complex of KD5 or AV7 peptides with H1N1 virus via nebulized inhalation significantly inhibited viral replication in infected mouse lung tissue, with peptide KD5 showing superior efficacy. These results not only validate the in vivo antiviral efficacy of the peptides but also provide crucial dosing parameters to support the development of nebulized inhalation-based post-exposure prophylaxis or early treatment strategies.

[0151] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0152] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0153] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A polypeptide with broad-spectrum antiviral activity, characterized in that, The amino acid sequence of the polypeptide includes: (a) One of SEQ ID NO: 1-SEQ ID NO: 10; or (b) A polypeptide derived from (a) having one or more amino acid substitutions, deletions, or additions, and possessing broad-spectrum antiviral activity, similar to the polypeptide shown in (a); or (c) is a polypeptide derived from (a) that has at least 85% sequence identity with the polypeptide shown in (a) and has broad-spectrum antiviral activity.

2. The polypeptide with broad-spectrum antiviral activity according to claim 1, characterized in that, The polypeptide includes pharmaceutically acceptable salts thereof, or polypeptide derivatives formed by modification of the N-terminus and / or C-terminus.

3. The polypeptide with broad-spectrum antiviral activity according to claim 2, characterized in that, The N-terminus and / or C-terminus are modified with one of the following: N-terminal acetylation, C-terminal amidation, linkage to a cell-penetrating peptide, linkage to a fatty acid chain, or linkage to a polyethylene glycol molecule.

4. The polypeptide with broad-spectrum antiviral activity according to claim 2, characterized in that, Pharmaceutically acceptable salts include trifluoroacetate, acetate, or hydrochloride.

5. The polypeptide with broad-spectrum antiviral activity according to claim 1, characterized in that, One or more of the amino acid sequences of the polypeptide are D-type amino acids.

6. A nucleic acid molecule, characterized in that: Encoding the polypeptide according to any one of claims 1-5.

7. A carrier, characterized in that: It includes the nucleic acid molecule as described in claim 6.

8. A host cell, characterized in that: It comprises the nucleic acid molecule of claim 6 or the vector of claim 7.

9. A pharmaceutical composition, characterized in that, The polypeptide comprising a therapeutically effective amount of any one of claims 1-5 and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9, characterized in that, The dosage form of the drug includes at least one of the following: intravenous injection, inhalation preparation, granules, tablets, pills, capsules, injections, and dispersants.

11. The pharmaceutical composition according to claim 10, characterized in that, The inhalation preparation is a nebulized inhalation solution, a nebulized inhalation suspension, or an inhalation dry powder.

12. A method for preparing the polypeptide according to any one of claims 1-5, characterized in that, The method includes: The crude product of the polypeptide was synthesized using a solid-phase synthesis method; The crude product was purified by reversed-phase high-performance liquid chromatography using a mobile phase system containing trifluoroacetic acid. The elution peak containing the target polypeptide was collected and then lyophilized to obtain the polypeptide.

13. Use of the polypeptide of any one of claims 1-5, or the pharmaceutical composition of any one of claims 9-11, in the preparation of a medicament for the prevention and / or treatment of viral infections.

14. The use according to claim 13, characterized in that, The working concentration of the peptide is 20 μM-50 μM.

15. The use according to claim 13, characterized in that, The viral infections include influenza A virus infection or lentivirus infection.

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