Polypeptide for inhibiting coronavirus replication and airborne transmission and application thereof

Abstract

The invention provides a polypeptide for inhibiting coronavirus replication and airborne transmission and application thereof. Specifically, the invention provides a branched peptide for inhibiting the replication and airborne transmission of coronavirus, the branched peptide comprises a plurality of peptide chain branches which take lysine as a connecting core and are coupled with the connecting core through amido bonds, and the sequence of the peptide chain branches is selected from the following groups: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or a combination of the SEQ ID NO: 1, the SEQ ID NO: 2 and the SEQ ID NO: 4, or a combination of the SEQ ID NO: 1, the SEQ ID NO: 2 and the SEQ ID NO: 4, or a combination of the SEQ ID NO: 1, the SEQ ID NO: 2 and the SEQ ID NO: 4. And the number of the peptide chain branches is greater than or equal to 2. The molecular peptide (especially the four-branch peptide) provided by the invention not only can inhibit the replication of the coronavirus, but also can efficiently block the interpersonal transmission of the high-transmissibility coronavirus.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to polypeptides that inhibit coronavirus replication and airborne transmission and their applications. Background Technology

[0002] With increased viral transmissibility and a surge in infections, reinfection with SARS-CoV-2 has become more frequent, exacerbating the risk of death and hospitalization. Furthermore, as SARS-CoV-2 spreads and evolves in the population, the basic reproduction number (R0) of the Delta and Omega variants has increased several times over. This increased transmissibility poses challenges to epidemic control measures. For influenza, studies have shown that Baxlovir can reduce influenza transmission. However, there are currently no similar drugs for SARS-CoV-2.

[0003] Preventing airborne transmission of SARS-CoV-2 is challenging because numerous factors influence the transmission of respiratory coronaviruses: viral replication capacity in the respiratory tract, the virus carried by asymptomatic and symptomatic individuals, and individual vaccination status all affect transmission. During the outbreak, widespread transmission accelerated evolutionary selection, leading to a continuous increase in the transmissibility of SARS-CoV-2 variants, with their basic reproduction number (R0) increasing from the original strain to the Delta and Omega-Jon variants. Currently known factors include human angiotensin-converting enzyme 2 (ACE2), the attachment factor heparan sulfate (a member of the glycosaminoglycan family), and other co-receptors, which play crucial roles in viral replication and transmission. However, these existing factors are insufficient to explain why the R0 of SARS-CoV-2 variants is higher than that of the original strain (strains preceding the alpha variant) and SARS-CoV-1.

[0004] Therefore, there is an urgent need in this field to develop an antiviral drug that can inhibit the replication of highly transmissible SARS-CoV-2 and block the airborne transmission of the virus, in order to reduce the harm caused by transmission and reinfection. Summary of the Invention

[0005] The purpose of this invention is to provide an antiviral drug that can inhibit the replication of highly transmissible SARS-CoV-2 and block the airborne transmission of the virus, so as to reduce the harm caused by transmission and reinfection.

[0006] In a first aspect of the present invention, a branched peptide for inhibiting coronavirus replication and airborne transmission is provided, the branched peptide comprising a plurality of peptide chain branches with lysine as a linker core and coupled to the linker core via amide bonds, wherein the sequences of the peptide chain branches are selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, or combinations thereof; Furthermore, the number of peptide chain branches is ≥2, preferably 2-10, and more preferably 4-8.

[0007] In another preferred embodiment, the sequence of each peptide branch is independently selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 4.

[0008] In another preferred embodiment, the coronavirus is SARS-CoV-2.

[0009] In another preferred embodiment, the SARS-CoV-2 is a highly transmissible variant selected from B.1.1.63, Delta (B.1.617.2), BA.2, or BA.5.

[0010] In another preferred embodiment, when the sequence of each peptide branch is SEQ ID NO: 1 and the number of peptide branches is 4, the branched peptide is a tetrabranched peptide 4PR26.

[0011] In another preferred embodiment, the structure of the tetrabranched peptide 4PR26 is shown in Formula I: (I).

[0012] In another preferred embodiment, when the sequence of each peptide branch is SEQ ID NO: 2 and the number of peptide branches is 4, the branched peptide is a tetrabranched peptide 4H22.

[0013] In another preferred embodiment, the structure of the tetrabranched peptide 4H22 is shown in formula V below: (V).

[0014] In another preferred embodiment, when the sequence of each peptide branch is SEQ ID NO: 4 and the number of peptide branches is 8, the branched peptide is an octapeptide 8P9R.

[0015] In another preferred embodiment, the structure of the eight-branched peptide 8P9R is shown in Formula II below: (II).

[0016] In another preferred embodiment, the branched peptide further comprises a branched peptide with a sequence as shown in SEQ ID NO: 3.

[0017] In another preferred embodiment, the branched peptide has an inhibition rate or blocking rate of at least 30% against the release of the coronavirus, more preferably 50%-100%, and even more preferably 75%-100%.

[0018] In another preferred embodiment, when the concentration of the branched peptide is 25 µg / mL, the inhibition rate of SARS-CoV-2 virus release is not less than 30%.

[0019] In a second aspect of the invention, a pharmaceutical composition is provided comprising a branched peptide as described in the first aspect of the invention and a pharmaceutically acceptable carrier.

[0020] In another preferred embodiment, the pharmaceutical composition is formulated as a dosage form suitable for nasal administration.

[0021] In another preferred embodiment, the dosage form of the pharmaceutical composition is a nasal drop, a nasal spray, or an inhaled powder.

[0022] In another preferred embodiment, the concentration of the branched peptide in the pharmaceutical composition is from 0.01 µg / mL to 100 mg / mL.

[0023] In a third aspect of the invention, the use of branched peptides as described in the first aspect of the invention or pharmaceutical compositions as described in the second aspect of the invention in the preparation of a medicament for inhibiting airborne transmission of coronaviruses is provided.

[0024] In another preferred embodiment, the drug is used for purposes selected from the group consisting of; (1) Used to inhibit coronavirus replication and airborne transmission; (2) To reduce the risk of individuals infected with coronavirus transmitting the virus through the air to susceptible individuals; (3) Used to prevent susceptible individuals from contracting coronaviruses through the air.

[0025] In another preferred embodiment, the drug is administered via nasal administration within 8 to 24 hours after the individual is infected with the coronavirus.

[0026] In another preferred embodiment, the coronavirus is SARS-CoV-2 virus or a variant thereof.

[0027] In another preferred embodiment, the coronavirus is a SARS-CoV-2 Delta (B.1.617.2) variant.

[0028] In a fourth aspect of the invention, a method for inhibiting airborne transmission of coronaviruses is provided, the method comprising administering to an individual in need an effective amount of a branched peptide as described in the first aspect of the invention or a pharmaceutical composition as described in the second aspect of the invention.

[0029] In another preferred embodiment, the application is used to reduce the risk of the individual being a source of infection.

[0030] In another preferred embodiment, the administration is performed via nasal administration within 8 to 24 hours after the individual is infected with the coronavirus.

[0031] In another preferred embodiment, the individual is a susceptible individual who is at risk of airborne exposure to coronavirus, and the application is for preventative protection.

[0032] In another preferred embodiment, the individual is an individual who has been infected with the coronavirus.

[0033] In another preferred embodiment, the individuals include humans and non-human mammals.

[0034] In another preferred embodiment, the non-human mammals include rodents and non-human primates.

[0035] In another preferred embodiment, the rodents include guinea pigs, mice, and rats.

[0036] In another preferred embodiment, the non-human primate includes monkeys.

[0037] In another preferred embodiment, the method is able to effectively block the airborne transmission of the coronavirus.

[0038] In another preferred embodiment, the method can significantly reduce the risk of an infected individual as a source of infection, wherein "significantly reduced" means compared to an infected individual who has not been treated by the method.

[0039] In another preferred embodiment, the method has an inhibition rate or blocking rate of not less than 30% against the release of the coronavirus, more preferably 50%-100%, and even more preferably 75%-100%.

[0040] In another preferred embodiment, the method is used to inhibit coronavirus replication and airborne transmission.

[0041] In a fifth aspect of the invention, a kit is provided, the kit comprising: (a) An effective amount of the branched peptide as described in the first aspect of the invention or the pharmaceutical composition as described in the second aspect of the invention; and (b) Instructions for use that indicate the use of the branched peptide or the pharmaceutical composition to inhibit the airborne transmission of coronaviruses.

[0042] In a sixth aspect of the present invention, a method for preparing a branched peptide that inhibits the airborne transmission of coronavirus is provided, the method comprising: coupling a linear polypeptide with a sequence selected from any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4 to an amino group of a lysine-linked core at the C-terminus of the linear polypeptide to form a branched peptide molecule having at least two (preferably two to ten, more preferably four to eight) peptide chain branches.

[0043] In another preferred embodiment, the coupling reaction forms a tetrabranched peptide with four peptide chain branches.

[0044] In a seventh aspect of the invention, a method for screening candidate branched peptides to determine whether they have the function of inhibiting airborne transmission of coronaviruses is provided, the method comprising the steps of: (s1) In the test group, coronaviruses and infected cells were treated in the presence of the test branch peptide, and data selected from the following groups were measured: (a) The amount of coronavirus attached to cells, A1; and / or (b) The amount of coronavirus released from infected cells, B1; In the control group, under identical conditions and without the test branched peptide, coronaviruses were treated with infected cells, and data from the control group selected from the following groups were measured: (c) The number of coronaviruses adhering to cells, A0; and / or (d) The amount of coronavirus released from infected cells, B0; and (s2) Compare the number of attachments and / or releases in the test group and the control group. If the number of attachments A1 in the test group is significantly higher than the number of attachments A0 in the control group, and / or the number of releases B1 in the test group is significantly higher than the number of releases B0 in the control group, then the above-mentioned branched peptide is a candidate branched peptide for inhibiting the airborne transmission function of coronavirus.

[0045] In another preferred embodiment, “significantly higher than” means A1 / A0 ≥ 30% and / or B1 / B0 ≥ 30%.

[0046] In an eighth aspect of the invention, a cell surface crosslinking agent comprising a branched peptide as described in the first aspect of the invention is provided for crosslinking coronavirus particles to the cell surface expressing glycosaminoglycans.

[0047] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0048] Figure 1 The diagram shows the charge analysis results of different SARS-CoV spike proteins in embodiments of the present invention. The top section displays a schematic diagram of the negative charge distribution of the spike proteins of SARS-CoV-1 (SARS-1), SARS-CoV-2 (Whhan-1), B.1.1.63, Delta variant, BA.2, and BA.5. The bottom section labels the corresponding number of negative charges (C) carried by each viral spike protein. The red areas in the diagram represent negatively charged surfaces, and the blue areas represent positively charged surfaces.

[0049] Figure 2 The figure shows the results of A549 cells failing to support SARS-CoV-2 replication in this embodiment of the invention, and the absence of ACE2 expression in these cells. Specifically, (a) RT-qPCR results show that SARS-CoV-2 replication efficiency is lower in A549 cells compared to VeroE6 cells; (b) Western blotting was used to detect the expression levels of ACE2 and tubulin in A549 and VeroE6 cells; VeroE6 cells express ACE2, but the presence of ACE2 protein was not detected in A549 cells.

[0050] Figure 3 The figure shown illustrates the virus attachment efficiency on A549 and Calu-3 cells in this embodiment of the invention. Different viral strains were incubated with A549(a) and Calu-3(b) cells at 4°C for 1 hour, and then the unattached viruses were removed. The amount of virus attached to the cell surface at 4°C was detected using RT-qPCR. Relative viral RNA (%) represents the normalized value of the attached virus amount versus the total virus amount used for attachment; * indicates P < 0.05, ** indicates P < 0.01, and data are expressed as mean ± standard deviation.

[0051] Figure 4 The figure shown illustrates the results of this invention's embodiments where P9RS did not show significant inhibitory effects against SARS-CoV-1 and SARS-CoV-2. VeroE6 cells were infected after treatment with P9RS (25 µg / ml) or DMEM (Mock), and plaque counts were recorded 3 days after infection. PFU (%) was defined as the percentage of plaque formation units in the P9RS-treated group relative to the untreated group, and data are expressed as mean ± standard deviation.

[0052] Figure 5The figure shown illustrates the effect of oscillation washing on virus adhesion in this embodiment of the invention. After treatment with DMEM or 4H30, the virus was incubated with A549 cells at 4°C. After removing unadhered viruses, the adhered viruses were washed using either a non-oscillating (DMEM group) or an oscillating (oscillating group) method. The viral RNA copy number adhering to the cells was then collected and measured; the relative viral RNA percentage was standardized using the DMEM treatment group as a baseline.

[0053] Figure 6 The figures show the virus titration results in the washing solution in this embodiment of the invention. After treatment with DMEM or 4H30, the virus was incubated with A549 cells at 4°C. After removing unadhered viruses, washing solutions under both non-shaking (DMEM group) and shaking (shaking group) conditions were collected, and the virus titer in the eluent was determined by a plaque assay. * indicates P < 0.05, ** indicates P < 0.01. Data are expressed as mean ± standard deviation and are derived from identified biological replicates.

[0054] Figure 7 The figures shown are related to the results of 4H30 promoting viral attachment on A549 and Calu-3 cells in the embodiments of the present invention. Different viral strains were treated with DMEM or 4H30 and then transferred to the surface of A549(a) and Calu-3(b) cells at 4°C and incubated for 1 hour. Then, the unattached viruses were removed. Viruses attached to the cell surface were detected by RT-qPCR.

[0055] Figure 8 The image shows the results of enhancing viral cell adhesion at 4°C using 4H30 in an embodiment of the present invention. The left image is a representative immunofluorescence image of the anti-spike protein (green) of the B.1.1.63 strain treated with DMEM or 4H30 attached to A549 cells. Mock: Uninfected cells (scale bar = 10 μm); the right image shows the quantitative analysis of the green fluorescence intensity of the viral spike protein. The green fluorescence intensity was calculated using 10 random microscopic fields. *** indicates P < 0.001; data are expressed as mean ± standard deviation.

[0056] Figure 9 The image shows the results of enhancing viral cell adhesion at 37°C using 4H30 in an embodiment of the present invention. Representative images show the attachment of virus-infected cells, treated with DMEM or 4H30, and labeled with Dio dye (green) to A549 cells. White arrows indicate cross-linked viral particles on the cell membrane. Mock: Uninfected cells (scale bar = 10 μm).

[0057] Figure 10The figures shown are the results of 4H30, 4H22, and 4PR26 significantly inhibiting SARS-CoV-2 infection in the embodiments of the present invention. Specifically, (a) 4H30 significantly inhibited SARS-CoV-2 infection of cells more than 2H30; (b) 4H22 and 4PR26 significantly inhibited SARS-CoV-2 infection of cells; and (c) 4PR26 and 4H22 were more effective than 4H30 in inhibiting the SARS-CoV-2 XBB mutant. SARS-CoV-2 was used to infect VeroE6 cells after treatment with different concentrations of peptides; the percentage of viral infection (%) was determined by calculating the number of plaques formed.

[0058] Figure 11 This figure shows the inhibition of delta variant release by 4H30 in an embodiment of the present invention. a and b are the results of virus detection in supernatant and cell lysate at 24 hours, respectively, using RT-qPCR. After cell infection with delta variant, uninfected cells were removed 1 hour after infection. Infected cells were treated with 4H30 or P9RS at 20 hours. Viral RNA in supernatant and cell lysate (n=4) was detected by RT-qPCR at 24 hours; * indicates P < 0.05, ** indicates P < 0.01; data are expressed as mean ± standard deviation of biological samples.

[0059] Figure 12 This invention illustrates how 4H22 and 4PR26 inhibit the release of SARS-CoV-2 in an embodiment of the present invention. After infecting cells with the XBB variant, uninfected cells were removed 6 hours after infection, and the infected cells were treated with either 4H22 or 4PR26. Viral RNA in the supernatant was detected at 10 hours using RT-qPCR (n=4); data are presented as mean ± standard deviation of biological samples.

[0060] Figure 13 This diagram illustrates the airborne transmission study protocol for infected hamsters, involving intranasal inoculation with PBS, 4H30, or remdesivir, as described in this invention. Hamsters were inoculated intranasally with the Delta strain (4 × 10⁻⁶). 3 TCID 50 Following infection, PBS, 4H30, or remdesivir were administered intranasally at 8 and 24 hours post-infection, respectively. At 24 hours, uninfected hamsters were placed in infected hamster cages, separated by plastic partitions to allow airborne transmission. After 6 hours of airborne transmission, the exposed hamsters were transferred to new cages for incubation. Lung and nasal turbinate tissues were collected on day 3 post-exposure, and viral load was determined using a plaque assay.

[0061] Figure 14 The results shown are the viral load measurements in the nasal cavity and lungs of infected hamsters in this embodiment of the invention (inoculation dose was 4 × 10⁻⁶). 3TCID 50 Infected hamsters were administered PBS, 4H30, and remdesivir (Remd, 0.2 μmol / kg) via intranasal instillation at 8 and 24 hours, followed by lung and nasal tissue collection at 30 hours to determine viral load.

[0062] Figure 15 The figure shows the viral load of the Delta variant strain in hamsters exposed in this embodiment of the invention. Uninfected hamsters were exposed to hamsters treated with PBS, 4H30, and remdesivir (Remd, 0.2 μmol / kg); after 6 hours of contact transmission, they were transferred to new cages for further culture. Lung and nasal tissues were collected after 72 hours to detect viral load. The dashed line indicates the detection limit.

[0063] Figure 16 The graph shows the airborne transmission efficiency of the virus in infected hamsters treated with different drugs in this embodiment of the invention. The airborne transmission rate of the Delta variant treated with PBS in the hamster model was 8 / 8. 4H3O (0.04 μmol / kg) blocked the transmission of the Delta variant among hamsters (blocking rate 6 / 8, transmission rate 2 / 8); while Remdesivir (Remd, 0.2 μmol / kg) only blocked transmission in 2 hamsters (blocking rate 2 / 8, transmission rate 6 / 8). Red hamsters represent infected individuals, and yellow hamsters represent uninfected individuals. * indicates P < 0.05.

[0064] Figure 17 This demonstrates that high concentrations of remdesivir significantly inhibited the replication and spread of the Delta variant in this embodiment of the invention. (a) Viral load in infected hamsters. (b) After treatment with remdesivir, no virus was detected in any of the contact hamsters exposed to the source of infection. Hamsters were nasally inoculated with the Delta strain (4 × 10⁻⁶). 3 TCID 50 Following infection, hamsters were treated intranasally with either PBS or remdesivir (Remd, 1.0 μmol / kg) at 8 and 24 hours post-infection, respectively. At 24 hours, uninfected hamsters were placed in the cages of infected hamsters in the same room, separated by plastic partitions to allow airborne transmission. Six hours after exposure, the exposed hamsters were transferred to new cages for incubation. Lung and nasal turbinate tissues were collected on day 3 post-exposure, and viral load was determined using a plaque assay. Detailed Implementation

[0065] Through extensive and in-depth research and numerous experimental screenings, the inventors discovered that branched peptides (4H22, 4PR26) can inhibit viral release by cross-linking viral spike proteins with cell surface glycosaminoglycans. The molecular peptides of this invention (especially the four-branched peptide) can both inhibit coronavirus replication and effectively block the human-to-human transmission of highly transmissible coronaviruses. Experiments have confirmed that the attachment efficiency of SARS-CoV-1 and SARS-CoV-2 variants (B.1.1.63, Delta, and Omicron) to respiratory epithelial cells gradually decreases, while the viral release efficiency increases. The branched peptides of this invention, by cross-linking viral particles with host cells, can both inhibit viral replication and block the airborne transmission route of highly transmissible SARS-CoV-2 variants. Based on this, the present invention was completed.

[0066] the term 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.

[0067] As used herein, “including” or “containing” includes “comprising,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0068] As used herein, the terms "tetrabranched peptide", "4-branched peptide", and "polypeptide" are used interchangeably and all refer to the polypeptide of the present invention that inhibits the replication and spread of coronavirus, namely, a linear peptide coupled to a 4-branched peptide by lysine residues at the C-terminus of the polypeptide; including: 4H30, 4H22 and 4PR26.

[0069] As used herein, the term "branched peptide" includes peptides with two or more branches, and the structural formulas of the branched peptides involved in this invention are shown in Table 1 below: Table 1 Among them, 4PR26 is a 4-branched peptide formed by coupling a linear peptide with a lysine residue at the C-terminus of the polypeptide with the sequence CWGPCPTAFRQIGNCGRFRVRCCRIR (SEQ ID NO:1). 4H22 is a 4-branched peptide formed by coupling a linear peptide to the C-terminus of the polypeptide with the sequence FCPRRYKQIGTCGLPGTKCCKK (SEQ ID NO:2) via a lysine residue. 4H30 is a 4-branched peptide formed by coupling a linear peptide to the C-terminus of the polypeptide with the sequence GAICHPVFCPRRYKQIGTCGLPGTKCCKKP (SEQ ID NO:3) via a lysine residue. 8P9R is an 8-branched peptide formed by coupling a linear peptide to the C-terminus of the polypeptide with the sequence NGAICWGPCPTAFRQIGNCGRFRVRCCRIR (SEQ ID NO:4) via a lysine residue. 2H30 is a two-branched peptide formed by coupling a linear peptide to the C-terminus of the polypeptide with the sequence GAICHPVFCPRRYKQIGTCGLPGTKCCKKP (SEQ ID NO:5) via a lysine residue.

[0070] In addition, the present invention uses linear peptide P9RS as a negative control, the sequence of which is NGAHSWHPNETHFRQIHNSGRHRVRSHRIR (SEQ ID NO:6).

[0071] Suppressing the spread of SARS-CoV-2 virus Preventing airborne transmission of SARS-CoV-2 is challenging because numerous factors influence the transmission of respiratory coronaviruses: viral replication capacity in the respiratory tract, the virus carried by asymptomatic and symptomatic individuals, and individual vaccination status all affect transmission. During outbreaks, widespread transmission accelerates evolutionary selection, leading to a continuous increase in the transmissibility of SARS-CoV-2 variants, with their basic reproduction number (R0) increasing from the original strain to the Delta and Omega-Jon variants. Currently known factors include human angiotensin-converting enzyme 2 (ACE2), the attachment factor heparan sulfate (a member of the glycosaminoglycan family), and other co-receptors, which play crucial roles in viral replication and transmission. However, these known factors are insufficient to explain why the R0 of SARS-CoV-2 variants is higher than that of the original strain (strains preceding the alpha variant) and SARS-CoV-1. Therefore, other potential factors must influence the transmissibility of SARS-CoV-2. Researching and identifying new factors could serve as potential targets for limiting viral transmission.

[0072] The main advantages of this invention include: (1) The branched peptides (4H22, 4PR26) provided by the present invention can inhibit viral release by crosslinking viral spike proteins with cell surface glycosaminoglycans.

[0073] (2) The branched peptide and its administration method provided by the present invention can effectively block the airborne transmission of highly transmissible coronaviruses (such as SARS-CoV-2 Delta variant) in animal models, with a blocking rate of over 75%, which is significantly better than the control antiviral drug remdesivir.

[0074] (3) This invention provides a novel drug strategy: by cross-linking viral particles with host cells, it can both inhibit viral replication and block the airborne transmission route of highly transmissible SARS-CoV-2 variants.

[0075] (4) The branched peptides (4H22, 4PR26) provided by this invention can be used to develop the first drug that can both inhibit coronavirus replication and effectively block the human-to-human transmission of highly contagious coronaviruses. It can not only treat patients infected with COVID-19, but also prevent the virus from spreading to healthcare workers or family members who have been in contact with the virus. This fills the gap in existing drugs that cannot inhibit viral release and transmission.

[0076] (5) The branched peptides 4H22, 4PR26 and 4H30 provided by the present invention have smaller molecular weights than branched peptide 4H30. Therefore, the synthesis cost of 4H22 and 4PR26 is reduced by more than 20%; and 4H22 and 4PR26 can more significantly inhibit XBB mutants.

[0077] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Unless otherwise specified, all experimental materials and reagents involved in this invention are commercially available.

[0078] Experimental methods: 1. Cell and virus culture A549 (CCL-185), VeroE6 (CRL-1586), VeroE6-TMPRSS2 (VeroE6-T), and Calu-3 (HTB-55) cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 IU / mL penicillin, and 100 μg / mL streptomycin. The viral strains used in this invention included SARS-CoV-1 and SARS-CoV-2 variants cultured in VeroE6 or VeroE6-T cells. Viral titers were determined using plaque assay and TCID50 method.

[0079] 2. Plaque Reduction Experiment This experiment used the plaque reduction assay to determine the antiviral activity of the peptides. The simplified steps are as follows: The peptides were dissolved in PBS buffer, and different concentrations (0–25.0 μg / mL) of peptides were premixed with SARS-CoV-2 at room temperature. After incubation for 45 minutes, the peptide-virus mixture was added to VeroE6 cells for infection. One hour after infection, the infection medium was removed, and 1% low-melting-point agarose was added to cover the cell layer. Two to three days post-infection, the cells were fixed with 4% formalin, stained with 0.1% crystal blue, and the number of plaques was counted.

[0080] 3. Viral RNA extraction and quantitative reverse transcription PCR (RT-qPCR) Viral RNA was extracted using a viral RNA mini extraction kit (QIAGEN, product number 52906, USA), and the RT-qPCR procedure followed previous methods. The extracted viral RNA was reverse transcribed into cDNA using the PrimeScript II first-strand cDNA synthesis kit (Takara, product number RR036A) and a GeneAmp® PCR system 9700 (Applied Biosystems, USA). The cDNA was then amplified using specific primers (Table 2) in a LightCycle® 480 SYBR Green I Master (Roche, USA) to detect SARS-CoV-1 and SARS-CoV-2. For quantification, standard plasmids were prepared in 10-fold serial dilutions (each reaction equivalent to 10...). 1 Up to 10 6 (Copy) to plot the standard curve. qPCR experiments were performed using a LightCycler® 96 system (Roche, USA).

[0081] Table 2 4. Virus attachment experiment A549 and Calu-3 cells were co-incubated with DMEM, 4H30, 4HB22, 4PR26 (25 μg / mL), or P9RS at room temperature for 30 minutes. After washing with PBS, SARS-CoV-1 and SARS-CoV-2 variants (MOI = 0.2) were added and the cells were incubated at 4°C for 1 hour. After removing unbound viruses, the amount of virus attached to the cell surface was detected by RT-qPCR. An equal volume of viral lysate was used as a normalization control. In the virus / cell pretreatment experiment, the virus or cells were pretreated with P9RS (25 μg / mL), followed by incubation with 4H30 at room temperature for 30 minutes. After one wash, the virus was added and the cells were incubated at 4°C for 1 hour. Unbound viruses were then washed with either shaking or non-shaking conditions, and the amount of virus attached to cells or in the supernatant was detected by RT-qPCR or plaque assay.

[0082] 5. Immunofluorescence detection After treating A549 cells with DMEM or 4H30 (25.0 μg / mL), SARS-CoV-2 (10 MOI) was added and the cells were infected at 4°C. Because the virus attachment efficiency is low at 4°C, the cells were co-incubated for two hours before washing, followed by cell membrane and nuclear staining at room temperature for 10 minutes. Cells were then fixed at room temperature for 1 hour, and then blocked with 5% BSA for 1 hour. Staining was performed sequentially using rabbit anti-spike protein antibody (Sino, product number 40590-T62, 1:6000) and goat anti-rabbit IgG Alexa-488 conjugate antibody (Life Technologies, product number A32731, 1:600) at room temperature for 45 minutes. In the virus release assay, VeroE6 cells were infected with the Delta variant (0.5 MOI), and 14 hours post-infection, peptides were added and the cells were incubated at 37°C for another 4 hours. Cells were fixed 18 hours post-infection and stained with mouse anti-spike protein IgG (Sino, product number 40592-MM57, 1:6000) and goat anti-rabbit IgG Alexa-488 conjugate antibody (Life Technologies, product number A32723, 1:600). All images were acquired using a confocal microscope (Carl Zeiss LSM 800, Germany).

[0083] 6. Virus release experiment Cells were infected (37°C) with SARS-CoV-1, SARS-CoV-2 original strains, and Delta variants (MOI = 0.1 or 0.01). Twenty hours post-infection, DMEM or a drug (peptide such as 4H30, at a concentration of 25.0 μg / mL) was added to the viral culture supernatant. Twenty-four hours post-infection, the supernatant and cell lysate were collected, and viral titers were detected by RT-qPCR. Each treatment group included 2-3 independent experiments, with biological replicates for each experiment.

[0084] 7. Hamster coronavirus transmission experiment Female hamsters (6-8 weeks old) were housed in a biosafety level 2 / 3 laboratory (ambient temperature 22-25°C, light-dark cycle), with free access to standard pelleted food and water. All experimental protocols followed approved standard operating procedures for biosafety level 2 / 3 animal facilities. Animal ethics guidelines were approved by the Animal Teaching and Research Ethics Committee of the University of Hong Kong (Approval No.: 5986-21).

[0085] To assess the transmissibility of the SARS-CoV-2 variant among hamsters, infected hamsters were first housed separately for 24 hours, then placed in a new cage with uninfected hamsters in between, separated by a perforated plastic partition (3 mm pores) to allow airborne transmission of the virus. Six hours after exposure, the uninfected hamsters were transferred to the new cage. Lung and nasal turbinate tissues were collected on day 3 post-exposure, and viral load was detected by RT-qPCR and plaque assay. For samples with viral loads close to the detection limit, a positive RT-qPCR and plaque assay result was considered a positive transmission event.

[0086] To evaluate the blocking effect of peptides such as 4H30 and 4H22 and remdesivir on viral transmission, hamsters were intranasally inoculated with SARS-CoV-2 (Delta) or SARS-CoV-1. Eight hours post-infection, either peptide (0.5 mg / kg = 0.04 μmol / kg) or remdesivir (1.0 μmol / kg or 0.2 μmol / kg) was administered intranasally. Twenty-four hours post-infection, one infected indicator hamster treated with the drug was housed with two uninfected contact hamsters, separated by a 3 mm perforated plastic partition, allowing airborne transmission of the virus. Six hours after exposure, lung and nasal turbinate tissues were collected from the infected hamster, and viral load was detected by RT-qPCR and plaque assay. The uninfected contact hamsters were transferred to new cages, and lung and nasal turbinate tissues were collected on day 3 post-exposure for viral load detection. For samples with viral loads close to the detection limit, a positive RT-qPCR and plaque assay result was considered a positive transmission event.

[0087] Example 1: Detection of the adhesion ability of Sabeclovirus to cells Based on the above experimental methods, this invention analyzed the spike protein charge of SARS-CoV-1 (2002), the prototype strain SARS-CoV-2 (2019, Wuhan-1), B.1.1.63 (D614G mutant), Delta, and the Omeprung variants BA.2 and BA.5, which have low transmissibility. Figure 1 Among them, Delta (B.1.617.2) and the Omecron variants BA.2 and BA.5 are SARS-CoV-2 variants.

[0088] The results showed that the SARS-CoV-1 spike protein carried a significantly higher negative charge (-23.92) than SARS-CoV-2. Notably, after three years of evolution in human hosts, the negative charge of the SARS-CoV-2 spike protein showed a gradual decreasing trend from the Wuhan strain (Wuhan-1, -11.96) to BA.5 (-4.94). Figure 1This decrease in the negative charge of the spike protein is consistent with epidemiological data showing that the basic reproduction number (R0) of SARS-CoV-2 increased from 3.0 to 38.0. The change in the spike protein's charge suggests that the virus has evolved to optimize its protein-level binding process.

[0089] This invention then examined the adhesion ability of these viruses to human respiratory epithelial cells Calu-3 (ACE2 positive) and A549 (ACE2 negative) at 4°C. A549 cells, lacking the ACE2 receptor, do not support SARS-CoV-2 replication. Figure 2 ab). Experimental results showed that the cell adhesion ability of the SARS-CoV-2 variant was significantly lower than that of SARS-CoV-1 (ab). Figure 3 This phenomenon (ab) is consistent with the decreasing trend of negative charge on the spike protein. The lower cell attachment efficiency of SARS-CoV-2 variants appears to be related to weakened charge interactions between the spike protein and the cell membrane. In contrast, the higher cell attachment efficiency of SARS-CoV-1 may stem from stronger charge interactions between its more negatively charged spike protein and the positively charged ion layer on the cell membrane surface. To further investigate the impact of charge interactions on viral attachment, this invention treated these sabella coronaviruses with the positively charged peptide P9RS, which lacks antiviral activity. Figure 4 As shown, P9RS does not inhibit SARS-CoV-1 and SARS-CoV-2 (D614G).

[0090] Since both viral attachment and release occur on the cell membrane surface, the virus's ability to attach to cells affects its release efficiency and transmissibility. This invention demonstrates experimentally that when washing cells with shaking, the attachment amount of Delta and BA.5 variants on A549 cells shows a more significant decrease compared to SARS-CoV-1 (SARS-CoV-1). Figure 5 This result is consistent with observations that the inherent attachment efficiency of Delta and BA.5 is lower than that of SARS-CoV-1. Figure 3 To verify this conclusion, the present invention further discovered that more delta and BA.5 virus particles could be detected in the eluent of samples treated with agitation and washing. Figure 6 These results indicate that strong viral adhesion to respiratory epithelial cells reduces viral release from infected cells.

[0091] Example 2: Branched peptides can broadly enhance the cell adhesion ability of sabevirus. To regulate viral attachment and thus block viral transmission, this invention first demonstrates that the positively charged polypeptide 4H30 can broadly enhance the attachment of SARS-CoV-1, B.1.1.63, Delta, BA.2, and BA.5 variants to A549 and Calu-3 cells. Figure 7 a-7b). This enhancement is independent of the ACE2 receptor, as A549 cells express almost no ACE2. (By 4°C) Figure 8 ) and 37°C ( Figure 9 Under these conditions, A549 cells were stained with the virus, further verifying the promoting effect of 4H30 on SARS-CoV-2 attachment—more virus particles were visible on the cell membrane after treatment with 4H30.

[0092] At the cellular level, 4H30 can cross-link SARS-CoV-2 to the cell surface by binding to glycosaminoglycans (GAGs). At the viral level, 4H30 can directly bind to the viral spike protein. Furthermore, 4H30 is more effective than 2H30 in inhibiting SARS-CoV-2 replication. Figure 10 a). Therefore, this invention further investigates the significant inhibitory effects of the four-branched peptides 4H22 and 4PR26 on viral infection ( Figure 10 b). Furthermore, this invention has found that, compared to 4H30, 4H22 and 4PR26 have a broader spectrum of inhibition against the SARS-CoV-2 virus. Figure 10 c); and 4H22 and 4PR26 were more effective at inhibiting the SARS-CoV-2 XBB variant (from the University of Hong Kong: PMID: 36493789), especially 4PR26, which completely infected XBB cells. These results indicate that the tetrabranched peptides can efficiently inhibit SARS-CoV-2 infection of cells.

[0093] Example 3: Branched peptides can significantly inhibit the release of sabevir from cells. Furthermore, when VeroE6 cells infected with the Delta variant were cultured for 20 hours (hpi), the addition of 4H30 or P9RS showed that 4H30 significantly reduced the amount of virus released in the supernatant. Figure 11 a), while simultaneously enhancing viral attachment to the cell surface ( Figure 11 b). Meanwhile, 4H22 and 4PR26 can effectively inhibit the release of Delta virus ( Figure 12 By capturing the virus and inhibiting its release, 4H30, 4H22, and 4PR26 have shown the potential to block the spread of SARS-CoV-2—since the virus particles with transmissible activity mainly originate from viral particles released extracellularly.

[0094] Example 4: Branched peptides can block airborne transmission of delta variants among hamsters. To investigate the blocking effect of 4H30 on the transmission of SARS-CoV-2, such as Figure 13 As shown, the present invention designs the following experiment: After inoculating with delta mutant strains (4 × 10⁻⁶), 3 TCID 50 Eight hours after primary infection, primary infected hamsters were treated intranasally with PBS, 4H30, or remdesivir (Remd), respectively. Twenty-four hours post-infection, uninfected hamsters and infected hamsters treated with PBS, 4H30, or remdesivir were placed in separate compartments within the same cage for airborne transmission experiments. Six hours after exposure, the contact group hamsters were transferred to a new cage for observation, and nasal and lung tissue samples were collected from the primary infected hamsters (Index hamsters) to determine viral load. Tissue samples from the contact group hamsters were collected 72 hours post-exposure.

[0095] Experimental results showed that 4H30 (0.04 μmol / kg) could inhibit the replication of the virus in the lungs of primary infected hamsters by up to 5 times, but had only a slight inhibitory effect on the replication of the virus in the nasal turbinate tissue. Figure 14 Remdesivir also inhibited viral replication in the lungs of animals (6-fold), but did not significantly inhibit viral replication in the nasal cavity. Data analysis from contact hamsters showed that... Figure 15 : 4H30 can significantly inhibit the airborne transmission of the virus, and no or very low viral loads were detected in the lungs or nasal cavity of hamsters that came into contact with it. From the analysis of transmission rate ( Figure 16 The infection rate in the PBS-treated group of exposed hamsters reached 100% (8 / 8). Notably, during airborne transmission, 4H30 successfully blocked 75% (6 / 8) of infections in exposed hamsters. Figure 16 This means the transmission blocking rate in the 4H30 treatment group was 6 / 8 (i.e., 75%). While remdesivir could inhibit the replication of the virus in the lungs of primary infected hamsters by 6-fold, its inhibitory effect on nasal turbinate virus replication was limited. Figure 14 ), blocking only 25% (2 / 8) of infections from contact with hamsters in airborne transmission ( Figure 16 Although high-dose remdesivir (1.0 μmol / kg) significantly inhibited viral replication and blocked transmission in primary infected hamsters ( Figure 17 However, under the same experimental conditions, its blocking efficiency is still lower than that of 4H30.

[0096] These results indicate that 4H30 is superior to remdesivir in blocking viral transmission, especially in primary infected hamsters with high viral loads. This enhancing effect may stem from the cross-linking activity of 4H30—it can capture viral particles and inhibit viral release. This transmission blocking ability has significant clinical value, particularly for patients in the middle to late stages of infection with high respiratory viral loads. In conclusion, viruses capable of airborne transmission must be released from cells. The four-branched peptides 4H30, 4H22, and 4PR26 effectively inhibit SARS-CoV-2 replication and release from infected cells. These data strongly support the possibility that these branched peptides, which inhibit viral replication and release, can effectively suppress airborne transmission of SARS-CoV-2.

[0097] discuss Compared to the less infectious SARS-CoV-1, the highly infectious SARS-CoV-2 variant has caused billions of infections. Investigating and elucidating the mechanisms behind this enhanced transmissibility is crucial for identifying targets to block SARS-CoV-2 transmission. This invention demonstrates that the spike protein of SARS-CoV-1 to SARS-CoV-2 carries a significantly reduced negative charge. This weakening of the negative charge is associated with reduced viral non-receptor attachment ability and enhanced transmissibility of the sabizione coronavirus. The electrostatic attraction between the negatively charged spike protein and the positively charged Stern layer may play a role in viral attachment and release. Based on this finding, this invention demonstrates that intranasal administration of the branched peptides of this invention, particularly the 4-branched peptide (such as 4H30, which can capture viral particles on host GAGs to inhibit viral release), effectively prevents the transmission of the SARS-CoV-2 Delta variant among hamsters. Intranasal administration offers multiple advantages in the prevention and treatment of respiratory viral diseases, including ease of self-administration, the ability to achieve high local drug concentrations, and reduced systemic side effects.

[0098] As SARS-CoV-2 spreads and evolves in the human population, the basic reproduction number (R0) of the Delta and Omega variants has increased several times over. This increased transmissibility poses challenges to epidemic prevention and control measures. In addition to non-pharmacological interventions that may affect the R value of SARS-CoV-2 transmission and existing antiviral drugs, this invention urgently aims to develop novel antiviral drugs targeting transmission determinants to block the spread of the SARS-CoV-2 virus.

[0099] This study confirms that the SARS-CoV-2 Omeprón and Delta variants, whose spike proteins carry less negative charge, exhibit significantly lower viral cell adhesion than SARS-CoV-1, but conversely, higher cell release efficiency. The low cell adhesion of these two highly transmissible variants may explain their shorter incubation periods and infectious viral shedding cycles compared to earlier variants and SARS-CoV-1. By using 4H30 to enhance viral non-receptor adhesion and inhibit viral release from cells, this invention successfully blocked airborne transmission of the Delta variant among hamsters. Notably, although both 4H30 and remdesivir showed less than a 10-fold inhibitory effect on viral replication in hamsters, 4H30 was significantly more efficient at blocking Delta transmission, confirming the importance of blocking viral release in reducing transmission risk. Compared to viral RNA replication inhibitors, this strategy has a significant advantage—in the later stages of infection, when high-titer viruses have been released into the respiratory tract, replication inhibitors cannot block the transmission of the released virus. Importantly, this mechanism has been validated in multiple ways: this invention has also verified that branched peptides 4H22 and 4PR26 can significantly inhibit viral release and prevent airborne transmission of the virus between hamsters.

[0100] This invention demonstrates that the electrostatic attraction between SARS-CoV-2 and cells can influence viral attachment and release processes, and that positively charged peptides (4H30, 4H22, and 4P9R26) possess dual functional activities—inhibiting viral replication and blocking viral release, thereby preventing SARS-CoV-2 transmission. This invention identifies a potential new target: blocking viral release and transmission by cross-linking the virus with host factors. This discovery opens up entirely new pathways for combating viral transmission. Given the ongoing prevalence of SARS-CoV-2 and its extremely high transmissibility leading to multiple waves of outbreaks, this invention urgently seeks drugs that can simultaneously and effectively inhibit viral replication and transmission to better control this highly infectious virus.

[0101] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A branched peptide that inhibits coronavirus replication and airborne transmission, characterized in that, The branched peptide comprises multiple peptide chain branches with lysine as the linker core and coupled to the linker core via amide bonds, wherein the sequences of the peptide chain branches are selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, or combinations thereof; Furthermore, the number of peptide chain branches is ≥2, preferably 2-10, and more preferably 4-8.

2. The branched peptide as described in claim 1, characterized in that, The coronavirus is SARS-CoV-2; the SARS-CoV-2 is a highly transmissible variant selected from B.1.1.63, Delta (B.1.617.2), BA.2 or BA.

5.

3. The branched peptide as described in claim 1, characterized in that, When the sequence of each peptide branch is SEQ ID NO: 1, and the number of peptide branches is 4, the branched peptide is a tetrabranched peptide 4PR26; the structure of the tetrabranched peptide 4PR26 is shown in Formula I below: （I）； When the sequence of each peptide branch is SEQ ID NO: 2, and the number of peptide branches is 4, the branched peptide is a tetrabranched peptide 4H22; the structure of the tetrabranched peptide 4H22 is shown in formula V below: （V）； When the sequence of each peptide branch is SEQ ID NO: 4, and the number of peptide branches is 8, the branched peptide is an octapeptide 8P9R; the structure of the octapeptide 8P9R is shown in Formula II below: (II)。 4. The branched peptide as described in claim 1, characterized in that, The sequence of the peptide branch also includes the sequence shown in SEQ ID NO:

3.

5. A pharmaceutical composition, characterized in that, It comprises the branched peptide as described in claim 1 and a pharmaceutically acceptable carrier.

6. Use of the branched peptide of claim 1 or the pharmaceutical composition of claim 5 in the preparation of a medicament for inhibiting airborne transmission of coronaviruses.

7. The use as described in claim 6, characterized in that, The drug is selected from the following groups of uses: (1) Used to inhibit coronavirus replication and airborne transmission; (2) To reduce the risk of individuals infected with coronavirus transmitting the virus through the air to susceptible individuals; (3) Used to prevent susceptible individuals from contracting coronaviruses through the air.

8. A reagent kit, characterized in that, The kit contains: (a) An effective amount of the branched peptide as claimed in claim 1 or the pharmaceutical composition as claimed in claim 5; and (b) Instructions for use that indicate the use of the branched peptide or the pharmaceutical composition to inhibit the airborne transmission of coronaviruses.

9. A method for screening candidate branched peptides to determine whether they have the function of inhibiting airborne transmission of coronaviruses, characterized in that, The method includes the following steps: (s1) In the test group, coronaviruses and infected cells were treated in the presence of the test branch peptide, and data selected from the following groups were measured: (a) The amount of coronavirus attached to cells, A1; and / or (b) The amount of coronavirus released from infected cells, B1; In the control group, under identical conditions and without the test branched peptide, coronaviruses were treated with infected cells, and data from the control group selected from the following groups were measured: (c) The number of coronaviruses adhering to cells, A0; and / or (d) The amount of coronavirus released from infected cells, B0; and (s2) Compare the number of attachments and / or releases in the test group and the control group. If the number of attachments A1 in the test group is significantly higher than the number of attachments A0 in the control group, and / or the number of releases B1 in the test group is significantly higher than the number of releases B0 in the control group, then the above-mentioned branched peptide is a candidate branched peptide for inhibiting the airborne transmission function of coronavirus.

10. A cell surface crosslinking agent comprising the branched peptide of claim 1, for crosslinking coronavirus particles to the cell surface expressing glycosaminoglycans.

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