Polypeptides and uses thereof in the prevention or treatment of flavivirus infections

Abstract

The present application relates to polypeptides and pharmaceutical compositions capable of specifically inhibiting flavivirus infection, such as Zika virus (ZIKV), Dengue virus (DENV) and Yellow fever virus (YFV). The polypeptides provided by the present application include Zika virus capsid protein or fragments thereof, which bind to Zika virus capsid protein dimers. This is particularly important for the treatment of flavivirus infection, especially the treatment of ZIKV infection in pregnant women, and can be used as a potential drug for the prevention or treatment of flavivirus infection, such as ZIKV, DENV and / or YFV.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and biomedicine, and relates to virus inhibitors, specifically polypeptides that inhibit Zika virus (ZIKV), dengue virus (DENV), and yellow fever virus (YFV) infection. Background Technology

[0002] Zika virus (ZIKV), like dengue virus (DENV) and yellow fever virus (YFV), belongs to the genus Flaviviridae in the family Flaviviridae. Its genome is a single-stranded positive RNA, about 10.8 kb in size, encoding three structural proteins (PrM, Envelope, Capside) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5).

[0003] ZIKV is a type of arbovirus. Although it is primarily transmitted through the bites of Aedes mosquitoes active during the day, studies have shown that it can also be transmitted from mother to child, sexually, and through blood transfusions. Reports have documented symptoms of ZIKV infection such as fever, rash, joint pain, muscle pain, headache, and conjunctivitis, but these symptoms are generally mild and self-limiting, typically improving within 2-7 days. However, it has been confirmed that ZIKV infection in pregnant women can penetrate the placental barrier, potentially causing microcephaly or other severe brain damage in infants, especially during the first trimester. Furthermore, ZIKV infection may also be associated with Guillain-Barré syndrome.

[0004] However, there are currently no effective vaccines or drugs for the prevention and treatment of ZIKV infection. Researchers in this field believe that the main measure to prevent ZIKV infection is to prevent mosquito bites, while the treatment of ZIKV infection is symptomatic, relieving the symptoms. Based on the current situation, there is an urgent need to study specific drugs for ZIKV, hoping to provide specific treatment for pregnant women infected with ZIKV in order to reduce the birth of microcephaly babies.

[0005] Because many flaviviruses share the same vector, the Aedes aegypti mosquito, there have been reports of co-infection with DENV, ZIKV, and other flaviviruses such as Chikungunya virus. However, pre-existing DENV antibodies in patients can enhance ZIKV infection through an antibody-dependent enhancement effect (ADE), where existing viral antibodies enhance the infection of a foreign virus. Similarly, some ZIKV antibodies can also enhance DENV infection. Modifying antibodies to reduce their binding to FcγR can decrease the ADE effect; however, this increases antibody production costs. Drugs with broad-spectrum anti-flavivirus activity could be used to treat cases of co-infection with ZIKV and DENV or other flaviviruses without considering the ADE effect. Currently, there are no effective vaccines or drugs for the prevention and treatment of ZIKV infection and its subsequent diseases caused by other flavivirus infections.

[0006] Given the high specificity and safety of peptide drugs, and their relatively short half-life in the human body, there is an urgent need for peptide drugs to target Zika virus infection and other flaviviruses such as DENV and YFV infection. Summary of the Invention

[0007] In response to Zika virus (ZIKV) infection and other flavivirus infections such as dengue virus (DENV) and yellow fever virus (YFV), this invention provides peptide drugs that can specifically inhibit flavivirus infections, especially peptide drugs that can specifically inhibit ZIKV, DENV and / or YFV infections. This is particularly important for the treatment of flavivirus infections, especially ZIKV infection in pregnant women, and can serve as a potential drug for treating ZIKV, DENV and / or YFV infections.

[0008] The above-mentioned objectives of the present invention are achieved through the following specific solutions.

[0009] In a first aspect, the present invention provides a polypeptide comprising a Zika virus capsid protein or a fragment thereof, said fragment being bound to a Zika virus capsid protein dimer.

[0010] In some embodiments, the fragment binds to the RNA-binding region of the Zika virus capsid protein and / or the envelope protein-membrane protein-binding region (EM-binding region).

[0011] In some implementations, the bonding is a non-covalent bond bonding.

[0012] In some embodiments, the polypeptide comprises the amino acid sequence shown in SEQ ID NO:1, or a conserved variant of the amino acid sequence shown in SEQ ID NO:1 obtained by adding, deleting, substituting or modifying one or more amino acids.

[0013] Another aspect of the present invention provides a fusion polypeptide comprising the polypeptide described in the first aspect and a membrane-penetrating peptide.

[0014] In some embodiments, the transmembrane peptide comprises an amino acid sequence represented by at least one of SEQ ID NOs:3 to 8.

[0015] In some embodiments, the transmembrane peptide comprises the amino acid sequence described in SEQ ID NO:3.

[0016] In some embodiments, the fusion protein comprises the amino acid sequence shown in SEQ ID NO:9, or a conserved variant of the amino acid sequence shown in SEQ ID NO:9 obtained by adding, deleting, substituting, or modifying one or more amino acids.

[0017] The polypeptides or fusion polypeptides of the present invention can be used to prevent, inhibit and / or treat infections of flaviviruses such as ZIKV, DENV and / or YFV.

[0018] Another aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide or fusion polypeptide described herein.

[0019] Another aspect of the present invention provides an expression vector comprising the nucleic acid molecule described herein.

[0020] Another aspect of the present invention provides a host cell comprising the nucleic acid molecule or expression vector described herein.

[0021] Another aspect of the present invention provides a pharmaceutical composition comprising the said polypeptide, or the said fusion polypeptide, or the said nucleic acid molecule, or the said expression vector, or the said host cell, and a pharmaceutically acceptable carrier.

[0022] In some embodiments, the pharmaceutical composition is in the form of tablets, powders, granules, pills, injections, suspensions, powders, emulsions, aerosols, gels, eye drops, sustained-release formulations, or sustained-release implants. In some embodiments, the pharmaceutical composition may be formulated as an injectable preparation. In some embodiments, the preparation is suitable for intravitreal injection, subcutaneous, intradermal, intramuscular, intravenous, intrathecal, or epidural administration.

[0023] The pharmaceutical compositions of the present invention are beneficial for the prevention, treatment or inhibition of ZIKV, DENV and YFV infection alone or in combination, and are particularly important for the treatment of ZIKV infection in pregnant women in epidemic areas.

[0024] Another aspect of the invention provides a medicine box comprising the pharmaceutical composition described herein, the pharmaceutical composition being encapsulated in a container, preferably a glass ampoule, glass bottle, plastic ampoule, plastic bottle, plastic bag, or pre-filled syringe. In some embodiments, the invention relates to a pharmaceutical unit dosage form suitable for parenteral administration to humans, the pharmaceutical unit dosage form comprising a pharmaceutical composition as described herein in a suitable container. In some embodiments, the suitable container is a pre-filled syringe. In some embodiments, the pre-filled syringe includes an injection needle.

[0025] Another aspect of the present invention provides the use of the polypeptide, the fusion polypeptide, the nucleic acid molecule, the expression vector, the host cell, the pharmaceutical composition, or the cassette described herein in the preparation of a medicament for the prevention, inhibition, or treatment of flavivirus infections.

[0026] In some embodiments, the flavivirus infection includes Zika virus (ZIKV), dengue virus (DENV), and / or yellow fever virus (YFV) infection.

[0027] In this invention, the polypeptide can be directly synthesized artificially or expressed in vitro through genetic engineering. Both the polypeptide and the nucleic acid molecule encoding it can be used to directly or indirectly obtain drugs that inhibit ZIKV and / or DENV and YFV infection. Therefore, the polypeptide and the nucleic acid molecule encoding it in this invention can be used to prepare anti-ZIKV drugs.

[0028] The peptides of this invention can effectively inhibit ZIKV and / or DENV and YFV infection by interfering with viral assembly. They have the characteristics of broad-spectrum inhibition of flaviviruses and low cytotoxicity. The peptide inhibitors of this invention are beneficial for the prevention, treatment or inhibition of ZIKV, DENV and YFV infection alone or in combination. They are especially important for the treatment of ZIKV in pregnant women in epidemic areas and can be used as potential drugs for the treatment of ZIKV and / or DENV and YFV infection. Attached Figure Description

[0029] Figure 1 This shows the location of peptide C3 in the ZIKV C protein, which consists of an EM-binding region, a dimerization region, and an RNA-binding region. C3 is located in the RNA-binding region of the C protein, which is involved in the assembly of viral nucleocapsid proteins and the viral genome.

[0030] Figure 2The results showed that peptide C3 could inhibit ZIKV-induced cytopathic effects, significantly inhibiting cytopathic effects in cell lines Vero E6 and BHK21, while the unrelated control peptide NR had no inhibitory effect.

[0031] Figure 3 The results showed that peptide C3 could inhibit the proliferation of ZIKV, with IC50 values ​​of approximately 0.593 μM and 0.2443 μM in the cell lines Vero E6 and BHK21, respectively, while the unrelated control peptide NR had no inhibitory effect.

[0032] Figure 4 The results showed that peptide C3 had no inhibitory activity against DENV-1, DENV-2, DENV-3, DENV-4, YFV, and JEV, indicating that peptide C3 has specific inhibition of ZIKV.

[0033] Figure 5 The results showed that peptide C3 had no significant inhibitory activity against CHIKV, indicating that peptide C3 has a certain species specificity.

[0034] Figure 6 The results showed that even at a concentration as high as 20 μM, peptide C3 did not exhibit significant cytotoxicity to BHK21, SF268, and Vero E6 cells, indicating that peptide C3 has low cytotoxicity and high safety.

[0035] Figure 7 The results showed that peptide C3 had no significant toxicity to pregnant mouse tissues at dosages up to 120 mg / kg, indicating that peptide C3 has no significant toxicity to pregnant mice.

[0036] Figure 8 The results showed that peptide C3 had no significant toxicity to offspring rat tissues at dosages up to 120 mg / kg, indicating that peptide C3 has no significant toxicity to offspring rats.

[0037] Figure 9 The study showed that 1 hour after intraperitoneal injection of ZIKV into A129 mice, intraperitoneal administration of the drug peptide C3 protected 40% of A129 mice from death caused by ZIKV infection (p = 0.0328, Log-rank test).

[0038] Figure 10 The interaction between C3 and the ZIKV C protein dimer is shown, with the action sites concentrated in the RNA-binding region and the EM-binding region of the ZIKV C protein. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0040] definition

[0041] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0042] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0043] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0044] The term "substitution" as used herein for amino acids refers to the replacement of at least one amino acid residue in an amino acid sequence with another different "substituted" amino acid residue. The term "insertion" as used herein for amino acids refers to the incorporation of at least one additional amino acid into an amino acid sequence. While inserts typically consist of one or two inserted amino acid residues, larger "peptide inserts" can also be prepared, for example, inserts of about three to five or even up to about ten, fifteen, or twenty amino acid residues. As disclosed above, the inserted residues can be naturally occurring or non-naturally occurring. The term "deletion" as used herein for amino acids refers to the removal of at least one amino acid residue from an amino acid sequence.

[0045] The peptides described herein may contain conserved amino acid substitutions at one or more amino acid residues, for example, at essential or non-essential amino acid residues. A “conserved amino acid substitution” is the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, in this document, essential or non-essential amino acid residues in the peptide are preferably replaced with another amino acid residue from the same side chain family. In some embodiments, amino acid segments may be replaced with segments that are structurally similar but differ in the order and / or composition of the side chain family members. Alternatively, in some embodiments, mutations may be randomly introduced along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants may be incorporated into the peptides of the present invention, and the ability of these peptides to bind to desired targets may be screened.

[0046] The term “cell-penetrating peptides (CPPs)” used in this article refers to a class of small molecule polypeptides composed of no more than 30 amino acids. Based on their amino acid composition, they can be divided into cationic cell-penetrating peptides and amphiphilic cell-penetrating peptides.

[0047] As used herein, the term "pharmaceuticalally acceptable carrier" refers to a component of a pharmaceutical preparation that is non-toxic to the subject, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0048] As used herein, the term "treatment" refers to the reduction and / or improvement of the disorder and / or related illness or symptoms, as well as the prevention of the worsening of disorder symptoms. Desired therapeutic effects include, but are not limited to, prevention of the onset or recurrence of the disease, symptom relief, reduction of any direct or indirect pathological outcome of the disease, prevention of metastasis, slowing of disease progression, improvement or relief of symptoms, and relief or improvement of prognosis. However, it should be understood that treating a disease or symptom does not require the complete elimination of the disease or related symptoms.

[0049] Zika virus (ZIKV), like dengue virus (DENV) and yellow fever virus (YFV), belongs to the genus Flaviviridae in the family Flaviviridae. Its genome is a single-stranded positive RNA, about 10.8 kb in size, encoding three structural proteins (PrM, Envelope, Capside) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5).

[0050] Dengue virus (DENV) belongs to a serotype subgroup within the genus Flaviviridae of the family Flaviviridae. There are four distinct but closely related serotypes (DENV-1, DENV-2, DENV-3, and DENV-4). Dengue virus is primarily transmitted by vector insects such as Aedes aegypti and Aedes albopictus, causing dengue fever, as well as dengue hemorrhagic fever and dengue shock syndrome, which have high morbidity and mortality rates. Dengue virus RNA contains approximately 11,000 nucleotides, with a mean molecular weight (MW) of 4.2 × 10⁻⁶. 6 It encodes 3 structural proteins (C, PrM, and E) and 7 non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5).

[0051] Yellow fever virus (YFV) is a single-stranded positive-sense RNA virus belonging to the genus *Flavavivirus* in the family Flaviviridae. The virus particle is spherical, 40-60 nm in diameter, with a lipid envelope and spikes on its surface; its genome length is approximately 11 kb. Yellow fever virus has only one serotype, but can be classified into multiple genotypes based on viral genome sequence characteristics. This virus can exhibit serological cross-reactivity with other viruses in the *Flavavivirus* genus, such as dengue virus, Zika virus, and West Nile virus.

[0052] This invention provides a polypeptide that can inhibit ZIKV infection. Experiments have shown that it has highly efficient inhibitory activity against ZIKV virus. At the same time, using ZIKV as the main model, its in vivo efficacy and toxicity were systematically studied, and the results showed that the polypeptide has good activity and safety.

[0053] This invention is based on the crystal structure of the ZIKV nucleocapsid (C) protein (PDB: 5Z0R, 5Z0V). The ZIKV C protein is truncated according to its functional domain, and experiments were conducted to study ZIKV peptide inhibitors. Results showed that the selected peptide (SEQ ID NO: 1) exhibited good inhibitory activity against ZIKV, inhibiting viral infection in the early stages. Animal experiments demonstrated that the peptide could inhibit vertical transmission of ZIKV in C57BL / 6 pregnant mice and protect A129 mice from death caused by ZIKV infection. Analysis of its mechanism of action revealed that it interacts with the C protein, inhibiting viral assembly and ultimately viral proliferation. Cytotoxicity testing showed that the peptide C3 was non-toxic to BHK21, SF268, and Vero cells.

[0054] In this invention, the polypeptide can be obtained by direct artificial synthesis or by in vitro expression through genetic engineering. Both the polypeptide and the gene can be used to directly or indirectly obtain drugs that inhibit ZIKV and / or DENV and YFV infection. Therefore, the polypeptide and the nucleic acid molecule encoding it in this invention can be used to prepare anti-ZIKV drugs.

[0055] This invention provides the use of peptides and nucleic acid molecules encoding them in the preparation of peptide inhibitors for ZIKV and / or DENV, YFV infection; the peptide inhibitors for inhibiting ZIKV and / or DENV, YFV infection have been shown in experiments to effectively inhibit ZIKV and / or DENV, YFV infection by interfering with viral assembly, and have the characteristics of broad-spectrum inhibition of flaviviruses and low cytotoxicity. The peptide inhibitors of this invention are beneficial for the treatment of ZIKV, DENV and YFV infection alone or in combination.

[0056] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0057] In the following embodiments, the method for determining nucleic acid copy number is as follows:

[0058] (1) ZIKV virus QRT-PCR nucleic acid detection was performed using ZIKV universal probe primers (for details on universal probe primers and plasmid templates, please refer to: Reference Liu Y, al. et. Nature. 2017 May);

[0059] (2) QRT-PCR was used to detect DENV-1, DENV-2, DENV-3, DENV-4, and YFV nucleic acids. The DENV and YFV detection primers used were all universal probe primers (for specific information on the probe primers for DENV-1, DENV-2, DENV-3, and DENV-4, please refer to: Santiago GA, al. et. PLoS Negl Trop Dis. 2013 Jul. For specific information on the probe primers for YFV, please refer to: Domingo C, al. et. J Clin Microbiol. 2012 Dec).

[0060] (3) QRT-PCR was used to detect CHIKV nucleic acid. The primers used were universal CHIKV probe primers (for details on CHIKV probe primers, please refer to: Pabbaraju K,al.et.J Clin Virol.2016Oct).

[0061] Example 1: C3 Design of Peptide Drugs

[0062] This embodiment is based on the crystal structure (PDB: 5Z0R, 5Z0V) of the ZIKV nucleocapsid protein (C) (Genebank No.: KX266255.1, SEQ ID NO: 1), and truncates the C protein according to its functional domains. Viral inhibition studies were conducted on peptide C3 (SEQ ID NO: 2, EAMEIIKKFKKDLAAMLRIINARKE), suggesting that it can inhibit the viral assembly stage. Figure 1 As shown, the results indicate that peptide C3 of the present invention is located in the RNA-binding region of the C protein, near the intermembrane region of the virus. Peptide C3 was synthesized by Genscript Biotech Co., Ltd.

[0063] Example 2: Detection of the inhibitory activity of peptide C3 on ZIKV-induced cytopathic effects

[0064] (1) A fusion protein containing polypeptide C3 and membrane-penetrating peptide (SEQ ID NO:9) and a fusion protein containing control polypeptide NR and membrane-penetrating peptide (SEQ ID NO:11) were synthesized respectively.

[0065] (2) The above fusion protein was serially diluted 2-fold in a 96-well plate using serum-free DMEM. The starting concentration was 20 μM, and a total of 7 concentrations were diluted. The drug volume in each well was 50 μL, and 3 replicates were set for each gradient.

[0066] (3) Dilute ZIKV with serum-free DMEM to a final concentration of 0.01 MOI. Add 50 μL to each well of the 96-well plate in (1) (wells with drug and virus are called drug wells). At the same time, set up a positive control group (with virus but no drug, i.e., virus wells), a negative control group (without virus and no drug, i.e., drug-free wells), and a control polypeptide group (with control polypeptide and virus, where the amino acid sequence of control polypeptide NR is shown in SEQ ID NO:10). Incubate the virus and drug at 37°C for 1.5 h.

[0067] (4) Add 100 μL of the mixture from (2) to a 96-well plate containing BHK21 cells or Vero E6 cells. Incubate at 37°C and 5% CO2 for 24 h, then replace the medium with DMEM medium containing 2% FBS.

[0068] (5) After 5-8 days, observe the cell state under a microscope and use CCK8 to detect the inhibitory activity of peptide C3 on ZIKV. Add 500 μL of CCK8 solution to 10 mL of serum-free DMEM (the amount used for one 96-well plate) and mix by inverting.

[0069] (6) Carefully aspirate the culture medium from the 96-well plate and add 100 μL of the reaction solution (4) to each well.

[0070] (7) After culturing at 37℃ for 2 hours, the OD 450 value was measured using an enzyme-linked immunosorbent assay (ELISA) reader;

[0071] (8) Calculate the inhibition rate of peptide C3 against viral infection. The formula is: peptide inhibition rate = (OD 450 value of drug well - average OD 450 value of viral well) × 100% / (average OD 450 value of no drug well - average OD 450 value of viral well).

[0072] The results are as follows Figure 2 As shown in the figure. The results indicate that the peptide C3 in this invention can inhibit ZIKV infection-induced cytopathic effects, significantly inhibiting cytopathic effects in the cell lines Vero E6 and BHK21, while the irrelevant control peptide NR has no inhibitory effect.

[0073] Example 3: Detection of the inhibitory activity of peptide C3 against ZIKV proliferation

[0074] (1) BHK21 cells and Vero E6 cells were seeded in 96-well plates, with 2 × 10⁶ cells per well. 4 Each sample is cultured at 37℃ and 5% CO2 for 12 hours before use.

[0075] (2) The fusion protein in Example 2 was serially diluted 10-fold in a 24-well plate using serum-free DMEM. The starting concentration was 20 μM, and a total of 7 concentrations were diluted. The drug volume in each well was 250 μL, and 3 replicates were set for each gradient.

[0076] (3) Dilute ZIKV with serum-free DMEM to a final concentration of 0.01 MOI. Add 250 μL to each well of the 24-well plate in (1) (the wells with drug and virus are called drug wells). At the same time, set up a positive control group (with virus but no drug, i.e., virus wells), a negative control group (without virus and no drug, i.e., no drug wells), and a control polypeptide group (with control polypeptide and virus). Incubate the virus and drug at 37°C for 1.5 h.

[0077] (4) Add 500 μL of the mixture in (2) to a 96-well plate containing BHK21 cells or Vero E6 cells;

[0078] (5) Incubate at 37℃ and 5% CO2 for 24 hours, then replace the culture medium with fresh DMEM containing 2% FBS.

[0079] (6) After 2-3 days, take 200 μL and use an automated nucleic acid extractor to extract ZIKV genomic nucleic acid;

[0080] (7) Use ZIKV universal probe primers to perform ZIKV virus QRT-PCR nucleic acid detection;

[0081] (8) Calculate the inhibition rate of peptide C3 against viral infection. The formula is: peptide inhibition rate = (ZIKV nucleic acid copy in virus well - ZIKV nucleic acid copy in drug well) × 100% / (ZIKV nucleic acid copy in virus well).

[0082] The results are as follows Figure 3 As shown in the figure. The results indicate that the peptide C3 in this invention can inhibit the proliferation of ZIKV, with IC50 values ​​of approximately 0.593 μM and 0.2443 μM in the cell lines Vero E6 and BHK21, respectively, while the irrelevant control peptide NR has no inhibitory effect.

[0083] Example 4: Detection of the inhibitory activity of peptide C3 against DENV-1, DENV-2, DENV-3, DENV-4, YFV, and JEV infections.

[0084] (1) BHK21 cells and Vero E6 cells were seeded in 24-well plates, with 1×10⁶ cells per well. 5 Each sample is cultured at 37℃ and 5% CO2 for 12 hours before use.

[0085] (2) The fusion protein in Example 2 was serially diluted 10-fold in a 24-well plate using serum-free DMEM. The initial concentration was 20 μM, with 7 dilutions. The drug volume per well was 250 μL, and each concentration was set up in 3 replicates.

[0086] (3) Dilute DENV-1, DENV-2, DENV-3, DENV-4, YFV, and JEV with serum-free DMEM to make the final concentration of the virus 0.01 MOI. Add 250 μL of each well to the 24-well plate in (1) (the wells with drugs and viruses are called drug wells). At the same time, set up a positive control group (with virus but no drug, i.e., virus wells) and a negative control group (without virus and no drug, i.e., no drug wells). Incubate the virus and drug at 37°C for 1.5 h.

[0087] (4) Add 500 μL of the mixture from (2) to a 24-well plate containing Vero E6 cells.

[0088] (5) Incubate at 37℃ and 5% CO2 for 24 hours, then replace the culture medium with fresh DMEM containing 2% FBS.

[0089] (6) After 2-3 days, take 200μL and use the pre-amplified nucleic acid extraction kit (Shenzhen Huayin Biotechnology Co., Ltd., specification 16T / plate, run program HY-216) to perform fully automated nucleic acid extraction of DENV-1, DENV-2, DENV-3, DENV-4, YFV17D, and JEV genomic nucleic acids;

[0090] (7) QRT-PCR was used to detect DENV-1, DENV-2, DENV-3, DENV-4, YFV, and JEV nucleic acids. The detection primers used for DENV, YFV, and JEV were all universal probe primers.

[0091] (8) Calculate the inhibition rate of peptide C3 against viral infection. The calculation formula is: peptide inhibition rate = (virus well nucleic acid copy - drug well nucleic acid copy) × 100% / (virus well nucleic acid copy);

[0092] The results are as follows Figure 4 As shown in the figure. The results indicate that peptide C3 has no inhibitory activity against DENV-1, DENV-2, DENV-3, DENV-4, YFV, and JEV, suggesting that peptide C3 in this invention has specificity in inhibiting ZIKV.

[0093] Example 5: Detection of the inhibitory activity of peptide C3 against CHIKV infection (Ahviridae virus).

[0094] (1) BHK21 cells and Vero E6 cells were seeded in 24-well plates, with 1×10⁶ cells per well. 5Each sample is cultured at 37℃ and 5% CO2 for 12 hours before use.

[0095] (2) The fusion protein in Example 2 was serially diluted 10-fold in a 24-well plate using serum-free DMEM. The initial concentration was 20 μM, with 7 dilutions. The drug volume per well was 250 μL, and each concentration was set up in 3 replicates.

[0096] (3) Dilute CHIKV with serum-free DMEM to a final concentration of 0.01 MOI. Add 250 μL to each well of the 24-well plate in (1) (the wells with added drug and virus are called drug wells). At the same time, set up a positive control group (with virus but no drug, i.e., virus wells) and a negative control group (without virus and no drug, i.e., no drug wells). Incubate the virus and drug at 37°C for 1.5 h.

[0097] (4) Add 500 μL of the mixture in (2) to a 24-well plate containing Vero E6 cells;

[0098] (5) Incubate at 37℃ and 5% CO2 for 24 hours, then replace the culture medium with fresh DMEM containing 2% FBS.

[0099] (6) After 2-3 days, take 200 μL and use an automated nucleic acid extractor to extract CHIKV genomic nucleic acid;

[0100] (7) QRT-PCR was used to detect CHIKV nucleic acid, and the primers used were CHIKV universal probe primers;

[0101] (8) Calculate the inhibition rate of peptide C3 against viral infection. The calculation formula is: peptide inhibition rate = (virus well nucleic acid copy - drug well nucleic acid copy) × 100% / (virus well nucleic acid copy);

[0102] The results are as follows Figure 5 As shown in the figure. The results indicate that peptide C3 has no significant inhibitory activity against CHIKV, suggesting that peptide C3 has a certain species specificity in inhibiting flaviviruses.

[0103] Example 6: Detection of the toxicity of peptide C3 to BHK21, Sf268, and Vero E6 cells.

[0104] (1) BHK21, Sf268 and Vero E6 cells were seeded into 96-well plates, with 1×10 cells per well. 4 Each sample should be incubated at 37℃ and 5% CO2 for 6 hours before use.

[0105] (2) The fusion protein in Example 2 was diluted with serum-free DMEM to obtain concentrations of 20 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM, and 0.0001 μM, 100 μL per well, with three replicates for each concentration, denoted as drug wells. Drug-free wells and cell-free wells (i.e., DMEM only) were also prepared.

[0106] (3) After culturing at 37℃ and 5% CO2 for 24 hours, observe the cell state under a microscope;

[0107] (4) Add 500 μL of CCK8 solution to 10 mL of serum-free DMEM and mix by inverting.

[0108] (5) Carefully aspirate the culture medium from the 96-well plate and add the reaction solution from (4) to each well, 100 μL;

[0109] (6) After culturing at 37℃ and 5% CO2 for 2 hours, the absorbance of each well was measured at OD 450 using an ELISA reader.

[0110] (7) Calculate cell viability using the formula: Cell viability = (OD 450 value of drug-treated wells - average OD 450 value of cell-free wells) × 100% / (average OD 450 value of drug-free wells - average OD 450 value of cell-free wells);

[0111] The results are as follows Figure 6 As shown in the figure. The results indicate that peptide C3 showed no significant toxicity to BHK21, Sf268, and Vero E6 at a concentration as high as 20 μM, suggesting that peptide C3 has low cytotoxicity and high safety.

[0112] Example 7: Safety test of peptide C3 in pregnant mice

[0113] (1) C57BL / 6 pregnant mice (12-14 days of gestation, n=20) were randomly divided into 4 groups. Blood samples were collected from the pregnant mice by orbital blood collection, and the contents of ALT (Alanine aminotransferase) and Creatinine were detected.

[0114] (2) Four groups were injected with different doses of the fusion protein in Example 2 (10 mg / kg, n=5; 35 mg / kg, n=5; 120 mg / kg, n=5), while the control group was injected with PBS via the tail vein (n=5).

[0115] (3) Blood samples were collected from pregnant mice by orbital blood collection 1 day, 3 days and 5 days after they were treated with the drug, and the contents of ALT (Alanine aminotransferase) and Creatinine were detected.

[0116] (4) The mice were then observed for 21 days, and the results were recorded.

[0117] (5) Two offspring mice and their corresponding mother mice were randomly selected for dissection and HE staining verification was performed;

[0118] (6) Statistical analysis was performed using GraphPad Prism Software. *, p<0.05; **, p<0.01; ***, p<0.001.

[0119] The results are as follows Figure 7 (pregnant mouse) and Figure 8 (Puppy mice) are shown. The results showed that, compared with the PBS control group, there were no lesions in the tissues of both mother mice and puppies in the peptide C3 treatment group, indicating that the mice had good safety even when the highest dose of C3 protein (120 mg / kg) was administered.

[0120] Example 8: Experiment on the lethal infection effect of peptide C3 against ZIKV in A129 mice.

[0121] (1) 1×10 5 PFU ZIKV (SMGC) was injected intraperitoneally into A129 mice (type I interferon deficiency, 4 weeks old, n=20);

[0122] (2) After 1 hour, the mice were randomly divided into two groups. One group was injected intraperitoneally with the fusion protein containing polypeptide C3 in Example 2 (10 mg / kg, n = 10), and the other group was injected intraperitoneally with an equal amount of the fusion protein (vehicle) containing NR polypeptide in Example 2 (10 mg / kg, n = 10).

[0123] (3) Inject once a day for 6 consecutive days;

[0124] (4) Two days after A129 mice were challenged with the virus, blood samples were collected from pregnant mice by orbital blood collection, and the viral load in the serum was determined by RT-qPCR.

[0125] (5) The mice were observed for 21 days, and the results were recorded;

[0126] (6) Statistical analysis was performed using GraphPad Prism Software. **, p<0.01;

[0127] The results are as follows Figure 9 As shown in the figure. The results showed that 1 hour after intraperitoneal injection of ZIKV into A129 mice, intraperitoneal administration of the drug peptide C3 could protect 40% of A129 mice from death caused by ZIKV infection (p = 0.0328, Log-rank test).

[0128] Example 9: Interaction site analysis of peptide C3 and ZIKV C protein

[0129] In this embodiment, ZDOCK 3.0.2 was used to predict the binding modes of peptide C3 and capsid protein-C, respectively. Before docking, the Alphafold2 model was used to predict the three-dimensional structures of capsid-protein-C and peptide C3. During docking, the default configuration of ZDOCK 3.0.2 was used for the study, performing global rigid stacking and scoring. Subsequently, the conformation with the best docking score was further refined using AMBER software, i.e., energy minimization optimization to remove local structural conflicts. Finally, PyMOL 2.5.2 was used for visualization analysis. The results are as follows: Figure 10 As shown in the figure, based on the docking prediction of the binding pattern between peptide C3 and the protein, the yellow dashed lines represent hydrogen bonding, the burgundy dashed lines represent salt bridging, and the burgundy solid short lines represent the amino acids in which the short peptide and protein interact. The results indicate that peptide C3 interacts with the ZIKV C protein dimer through non-covalent binding, and its binding sites are concentrated in the ZIKV C protein RNA-binding region and EM-binding region.

[0130] Information on some of the sequences involved in this invention is provided in Table 1 below.

[0131] Table 1

[0132]

[0133] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A polypeptide comprising a Zika virus capsid protein or a fragment thereof, said fragment being bound to a Zika virus capsid protein dimer, said polypeptide having the amino acid sequence shown in SEQ ID NO:

2.

2. The polypeptide according to claim 1, characterized in that, The fragment binds to the RNA-binding region of the Zika virus capsid protein and / or the envelope protein-membrane protein-binding region.

3. A fusion polypeptide comprising the polypeptide of claim 1 or 2 and a membrane-penetrating peptide, wherein the amino acid sequence of the fusion polypeptide is shown in SEQ ID NO:

9.

4. A nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide of claim 1 or 2 or the fusion polypeptide of claim 3.

5. An expression vector comprising the nucleic acid molecule of claim 4.

6. A host cell comprising the nucleic acid molecule of claim 4 or the expression vector of claim 5.

7. A pharmaceutical composition comprising the polypeptide of claim 1 or 2, or the fusion polypeptide of claim 3, or the nucleic acid molecule of claim 4, or the expression vector of claim 5, or the host cell of claim 6, and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition is in the form of tablets, powders, granules, pills, or powder.

9. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition is in the form of a suspension, emulsion, or eye drops.

10. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition is in the form of an aerosol.

11. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition is in the form of a gel.

12. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition is in the form of an injectable preparation.

13. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition is in the form of a sustained-release agent or a sustained-release implant.

14. The use of the polypeptide of claim 1 or 2, the fusion polypeptide of claim 3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the host cell of claim 6, or the pharmaceutical composition of any one of claims 7-13 in the preparation of a medicament for the prevention, inhibition, or treatment of flavivirus infections.

15. The application according to claim 14, characterized in that, The Flavivir infections include Zika virus (ZIKV), dengue virus (DENV), and / or yellow fever virus (YFV) infections.

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