Inactivated coronavirus fusion protein and uses thereof

Abstract

Provided are fusion proteins that inactivate coronaviruses and uses thereof. The fusion proteins herein comprise an ACE2 peptidase domain comprising the amino acid sequence set forth in SEQ ID NO: 1 directly or via a linker connected to a coronavirus polypeptide therapeutic that targets the HR1 domain. The fusion proteins are broad-spectrum, highly potent, and can inactivate viruses at an early, free stage.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a protein that inactivates SARS-CoV-2 and other coronaviruses that use ACE2 as a cell receptor, and its applications. Background Technology

[0002] The COVID-19 pandemic is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). This virus uses the same cellular receptor as SARS-CoV-2, but it is more transmissible. As the virus spreads, infects more people, and the infection rate increases, many variants have emerged from the original strain. Following the WHO's designation of variants of concern (VOCs) such as B.1.1.7 (first discovered in the UK, α), B.1.351 (first discovered in South Africa, β), P.1 (first discovered in Brazil, γ), B.1.617 (first discovered in India, δ), and B.1.529 (first discovered in South Africa, Omicron), Omicron has further evolved into different subtypes (BA.1–BA.5, BF.7, BQ.1, XBB, EG.5, etc.). Although the current variant strains cause relatively mild symptoms in adults, the increased transmissibility and immune evasion capabilities resulting from viral evolution have reduced the effectiveness of previously marketed vaccines and antibodies, increasing the chance of reinfection and posing a safety risk to human health. Based on this situation, there is an urgent need to develop a broad-spectrum, highly effective antiviral agent designed based on conserved viral targets that could potentially eliminate the adverse effects of viral infection.

[0003] SARS-CoV-2 infects target cells via its spike protein (S), which is a homotrimer composed of two subunits, S1 and S2. The receptor-binding domain (RBD) of the S1 subunit interacts with the cellular receptor ACE2. Following cleavage by the host protease, the S2 subunit undergoes a conformational change, with the N-terminal fusion peptide inserting into the target cell membrane, exposing the N-terminal heptapeptide repeat region (NHR or HR1) trimer. Subsequently, three C-terminal heptapeptide repeat regions (CHR or HR2) are inserted antiparallel into the hydrophobic grooves of the HR1 trimer, forming a 6-helix bundle (6-HB). This bundle brings the viral envelope closer to the target cell membrane, mediating membrane fusion. Viral genetic material then enters the target cell through the fusion pore to replicate and produce new viral particles.

[0004] In the development of broad-spectrum coronavirus entry inhibitors, the helical region of the S2 protein is an important target. S2P6 is an antibody that targets the stem-helical region of the SARS-CoV-2 S2 protein. It has broad-spectrum neutralizing activity against MERS-CoV, SARS-CoV, HCoV-OC43, 229E, etc., but its IC50 (half-maximal inhibitory concentration) is high and its activity is not good.

[0005] Chinese patent application CN107022008A discloses a modified three polypeptides (including the EK1 polypeptide) derived from HCoV-OC43HR2. These are broad-spectrum and highly effective coronavirus entry inhibitors that can bind to the HR1 trimer exposed by the coronavirus after receptor stimulation, thereby competitively inhibiting the binding of the virus's own HR2, and thus inhibiting the formation of fusion pores to prevent the virus from entering target cells.

[0006] Chinese patent application CN115057914A discloses EK1 stapling and alanine modification. Antiviral activity tests have demonstrated that this type of polypeptide molecule has anti-COVID-19 activity and has potential value in the COVID-19 pandemic.

[0007] There remains an urgent need in this field to develop a broad-spectrum, highly effective drug that can inactivate the virus in its early, free stage. Summary of the Invention

[0008] This invention is partly based on the inventors' findings that currently developed peptide and protein drugs, or small molecule compounds based on virus replication-related enzymes, are unable to inactivate viruses and struggle to exert inhibitory effects during the free phase before viral attachment to cells, potentially leading to failure to block viral infection in its initial stages. While coronavirus peptide therapeutics targeting the HR1 domain, such as EK1 peptide, can efficiently inhibit coronavirus infection, they cannot inhibit the virus during its free phase before attachment to cells, which may reduce drug utilization and limit the drug's effectiveness. Therefore, there is an urgent need to develop a broad-spectrum, highly effective drug that can inactivate the virus in its early free phase. The inventors discovered that interacting a cell membrane surface receptor analog (free ACE2 protein) with the S1 subunit of the viral envelope protein can promote a conformational change in the S2 subunit, exposing the binding sites of coronavirus peptide therapeutics targeting the HR1 domain, such as EK1 peptide. Therefore, the conjugate proteins of ACE2 protein and coronavirus peptide therapeutics targeting the HR1 domain, such as EK1 peptide, possess the feasibility of being highly effective viral inactivation drugs.

[0009] This invention is mainly based on the following insights:

[0010] The inventors conducted in-depth research on this topic. Previous studies revealed that secretory ACE2 present in the human body can act as an ACE2 analog on the cell membrane surface, blocking the RBD in the viral S protein to inhibit viral infection. Based on this, it was discovered for the first time that the secretory ACE2 peptidase-binding domain can induce conformational changes in the S protein to promote the binding of HR1 target inhibitors such as the EK1 peptide (which was considered impossible in the traditional understanding of single-receptor-mediated viral infection). Based on this novel discovery of secretory receptor-induced viral protein conformational changes, the inventors creatively designed a dual-target viral infection inhibitor protein coupled (e.g., through a linker) between the ACE2 peptidase-binding domain and the HR1 target inhibitor, such as the EK1 peptide. Since the conformational change induced by the ACE2 peptidase-binding domain alone is unstable and may pose a certain risk of promoting viral infection, the coupled HR1 target inhibitor, such as the EK1 peptide, can bind to the HR1 target exposed after the S protein conformational change, further stabilizing the S protein structure and thus completely eliminating the virus's ability to reinfect cells. Therefore, the fusion protein designed in this invention, in addition to its traditional viral infection inhibitory effect, also has the effect of directly inactivating free viruses.

[0011] In one aspect, a fusion protein is provided comprising an ACE2 peptidase domain directly or via a linker and a coronavirus polypeptide therapeutic agent targeting an HR1 domain, wherein the ACE2 peptidase domain comprises (1) the amino acid sequence shown in SEQ ID NO:1; (2) an amino acid sequence having 80%, 85%, 90%, 95%, 98%, or 99% or more identity with the amino acid sequence shown in SEQ ID NO:1; or (3) an amino acid sequence in which one or more amino acid residues are mutated in the amino acid sequence shown in SEQ ID NO:1.

[0012] SEQ ID NO:1

[0013]

[0014] In one embodiment, the coronavirus peptide therapeutic agent comprises a peptide or a derivative thereof selected from the group consisting of: SEQ ID NO:2 (SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL), SEQ ID NO:12 (SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKELGSGGRRRRRR); and SEQ ID NO:6 (NPGDINVTFLDLEYEMKKLEEAIKKLEESYIDLKKL) or an amino acid sequence having more than 80%, 85%, 90%, 95%, 98%, or 99% identity with any one of SEQ ID NO:2, 12, and 6.

[0015] In one implementation, the derivative is a polyethylene glycol-modified or cholesterol-modified derivative of a polypeptide.

[0016] In one implementation, the mutation is selected from substitution, addition, insertion, or deletion.

[0017] In one implementation, the substitution is a conserved amino acid substitution.

[0018] In one embodiment, the joint is a flexible joint, preferably (G). n1 (SGG) n2 (SGGG) n3 (GGSGG) n4 (GSSGG) n5 Or (GGGGS) n6 Where n1 = 5-35, 10-30, or 20-25; n2 = 1-13, 2-10, or 3-8; n4, n5, or n6 = 1-10, 2-7, or 3-6, for example, n4, n5, or n6 = 5. In one embodiment, the connector is (GGGGS). n6 For example, n6 can be 1, 2, 3, 4, 5, 6, or 7.

[0019] In one embodiment, the ACE2 peptidase domain is located at the N-terminus of the fusion protein and the coronavirus peptide therapeutic agent is located at the C-terminus of the fusion protein.

[0020] In one embodiment, the fusion protein includes a portion with an extended half-life, such as an IgG Fc-binding peptide, an amino acid sequence such as SEQ ID NO:13 (DCAWHLGELVWCT), or a variant thereof with substitutions, insertions, deletions, or additions of one or more amino acids.

[0021] In one embodiment, the fusion protein comprises an ACE2 peptidase domain-first adapter-EK1 polypeptide or an EK1 polypeptide-first adapter-ACE2 peptidase domain, and the IgG Fc binding peptide is directly or via a third adapter linked to the ACE2 peptidase domain or the EK1 polypeptide.

[0022] In one implementation, the third connector is (G). m1 (SGG) m2 (SGGG) m3 (GGSGG) m4 (GSSGG) m5 Or (GGGGS) m6 Where m1 = 5-35, 10-30 or 20-25; m2 = 1-13, 2-10 or 3-8; m4, m5 or m6 = 1-10, 2-7 or 3-6, for example m4, m5 or m6 = 5.

[0023] In one embodiment, the fusion protein comprises the amino acid sequence shown in SEQ ID NO:7.

[0024] SEQ ID NO:7:

[0025]

[0026]

[0027] In one embodiment, the C-terminus of the fusion protein further includes an enzyme cleavage site and / or a purification tag.

[0028] In one implementation, the restriction site is the HRV 3C restriction site.

[0029] In one implementation, the purification tag is a His tag, such as an 8xHis tag.

[0030] In one embodiment, the fusion protein comprises the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:7.

[0031] On the other hand, nucleic acids are provided, which encode the fusion protein according to this article.

[0032] In one implementation, the nucleic acid comprises a codon-optimized nucleotide sequence.

[0033] In one implementation, the nucleotide sequence is a codon-optimized nucleotide sequence for expression in eukaryotic or prokaryotic cells, such as yeast or Escherichia coli.

[0034] In another aspect, a carrier containing the nucleic acid of this article is provided.

[0035] In one embodiment, the vector comprises an expression control element operatively linked to the nucleic acid, a purified tag nucleotide, and / or a leader sequence nucleotide.

[0036] In one implementation, the expression control element is selected from promoters, terminators, or enhancers.

[0037] In one implementation, the purification tag is selected from His tag, GST tag, MBP tag, SUMO tag, or NusA tag.

[0038] In one implementation, the vector is an expression vector, cloning vector, shuttle vector, or viral vector, such as a plasmid or granule. For example, the vector is a pFuse-hIgG1-Fc2 series plasmid or a pET series plasmid.

[0039] In another aspect, a host cell is provided, which contains the nucleic acids described herein or the vectors described herein. Preferably, the host cell is a eukaryotic or prokaryotic cell. Preferably, the eukaryotic cell is a yeast cell, animal cell, and / or insect cell, and / or the prokaryotic cell is an Escherichia coli cell, such as Escherichia coli BL21 cell.

[0040] In another aspect, compositions are provided comprising one or more of the fusion proteins, nucleic acids, vectors, and host cells described herein. Preferably, the composition is a pharmaceutical composition or kit. Preferably, the composition comprises a pharmaceutically acceptable carrier, excipient, or diluent. Preferably, the composition is a solid, liquid, or gel composition. Preferably, the composition is an injectable composition or an oral composition. Preferably, the composition is in the form of tablets, capsules, drops, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, suppositories, or lyophilized powders. Preferably, the composition further comprises another therapeutic agent against coronaviruses. Preferably, the therapeutic agent is selected from Paxlovid, abavirin / romisvirimilab, COVID-19 human immunoglobulin, convalescent plasma, glucocorticoids, and interleukin-6 inhibitors.

[0041] In another aspect, a method for preparing the fusion protein described herein is provided, comprising:

[0042] (1) Culture the host cells as described herein under suitable culture conditions;

[0043] (2) Harvesting host cells and / or culture medium containing the fusion protein; and

[0044] (3) Purify the fusion protein.

[0045] Preferably, step (3) includes separating the fusion protein using column separation with a ligand targeting the purification tag when the fusion protein contains a purification tag, and / or removing the purification tag by enzymatic cleavage using a tool enzyme. Preferably, the purification tag is a histidine tag, such as an 8xHis tag. Preferably, the column separation is nickel column separation. Preferably, when the fusion protein contains an HRV 3C cleavage site, the tool enzyme is an HRV 3C enzyme. Preferably, the method further includes a step of modifying the fusion protein. Preferably, when the ACE2 peptidase domain is at the N-terminus of the fusion protein and the coronavirus peptide therapeutic agent is at the C-terminus of the fusion protein, the C-terminus of the coronavirus peptide therapeutic agent is PEGylated or a cholesterol moiety is added.

[0046] In another aspect, the use of the fusion protein, nucleic acid, vector, host cell, and / or composition described herein in the preparation of a medicament or kit for treating or preventing coronavirus infection or coronavirus infection-related disease in a subject is provided. Methods for treating or preventing coronavirus infection or coronavirus infection-related disease in a subject are provided, including administration of the fusion protein, nucleic acid, and / or composition described herein. Preferably, the coronavirus contains ACE2 as a cell receptor. Preferably, the coronavirus is one or more of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), HCoV, RsSHC014-CoV, or RsW1V1-CoV. Preferably, HCoV is selected from HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1 strains. Preferably, the SARS-CoV-2 strain is selected from the prototype, Alpha, Beta, Gamma, Delta, Lambda, BA.1, BA.2, BA.2.2, BA.2.9, BA.2.12.1, BA.2.75, BA.3, BA.4.6, BA.5, BF.7, XBB, or BQ.1 viral strains. Preferably, the drug is in the form of tablets, capsules, drops, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal formulations, suppositories, or lyophilized powder. Preferably, the subject is a human or mammal, such as a companion animal, zoo animal, or livestock.

[0047] In another aspect, a method for treating coronavirus infection for non-therapeutic purposes is provided, comprising contacting coronavirus-infected cells with the fusion protein and / or composition described herein; or contacting uninfected cells with the fusion protein and / or composition described herein to avoid coronavirus infection. Preferably, the coronavirus contains ACE2 as a cell receptor. Preferably, the coronavirus is one or more of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), HCoV, RsSHC014-CoV, or RsW1V1-CoV. Preferably, HCoV is selected from HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1 strains. Preferably, SARS-CoV-2 is selected from the prototype, Alpha, Beta, Gamma, Delta, Lambda, BA.1, BA.2, BA.2.2, BA.2.9, BA.2.12.1, BA.2.75, BA.3, BA.4.6, BA.5, BF.7, XBB, or BQ.1 viral strains.

[0048] In another aspect, methods are provided for inhibiting coronavirus growth in vitro, preventing or inhibiting coronavirus infection of cells in vitro, said methods comprising the step of contacting the fusion protein and / or composition described herein with an article or sample containing or at risk of containing coronavirus. Methods for preventing and / or treating coronavirus infection are provided. Preferably, the coronavirus contains ACE2 as a cell receptor. Preferably, the coronavirus is one or more of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), HCoV, RsSHC014-CoV, or RsW1V1-CoV. Preferably, HCoV is selected from HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1 strains. Preferably, SARS-CoV-2 is selected from the prototype, Alpha, Beta, Gamma, Delta, Lambda, BA.1, BA.2, BA.2.2, BA.2.9, BA.2.12.1, BA.2.75, BA.3, BA.4.6, BA.5, BF.7, XBB, or BQ.1 viral strains.

[0049] The advantages of this invention include:

[0050] 1. The fusion protein of the present invention can induce a conformational change in the S2 subunit by binding to the RBD in the S1 subunit of the spike protein of SARS-CoV-2 and other coronaviruses that use ACE2 as a cell receptor through the ACE2 functional domain. Furthermore, it can cause free virus to lose its infectivity by binding to the exposed HR1 target in the S2 subunit through the EK1 peptide, thereby terminating the viral infection process from the initial stage. This greatly improves the drug utilization rate compared to other viral protease inhibitors and entry inhibitors.

[0051] 2. By using a protein-peptide conjugation strategy, the inhibitory activity of ACE2 protein can be increased by 26 to 600 times, which will effectively reduce costs in the later stages of drug development; at the same time, the EK1 peptide, which only has antiviral activity, can produce a virus inactivation effect.

[0052] 3. In vivo mouse model tests show that the protein of the present invention, as an inhibitor or inactivator, has high safety and good drug development potential.

[0053] 4. The inventors discovered that the linker length used in the fusion protein affects the inhibitory or inactivating properties of the fusion protein against coronaviruses, with the AL5E protein exhibiting the best inhibitory or inactivating properties. Attached Figure Description

[0054] Figure 1Results of gel filtration chromatography and Coomassie brilliant blue staining for identification of AL2E, AL5E, AL6E, and AL7E proteins.

[0055] Figure 2 The results of pseudovirus inhibition screening for SARS-CoV-2 VOC and Omicron mutant strains by using AL2E, AL5E, AL6E, and AL7E proteins.

[0056] Figure 3 The results show the inhibitory effect of AL5E protein on the infection of Omicron mutant strain BA.2.2 subtype live virus.

[0057] Figure 4 Results of AL5E protein inactivation of SARS-CoV-2VOC and Omicron mutant pseudovirus.

[0058] Figure 5 Results of inhibition and inactivation of AL5E protein and other coronaviruses using ACE2 as a cell receptor.

[0059] Figure 6 Results of AL5E protein prevention and protection of K18-ACE2 transgenic mice from human coronavirus NL63 infection.

[0060] Figure 7 The results of using AL5E protein to inactivate human coronavirus NL63 to protect K18-ACE2 transgenic mice from infection. Detailed Implementation

[0061] 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. Unless otherwise specified, all units used in this specification are International Standard Units (SI), and all numerical values ​​and ranges appearing in this invention should be understood to include unavoidable systematic errors. Embodiments of the invention are described below, but the invention is not limited thereto.

[0062] In this invention, the terms "comprising," "having," "including," or "containing" can mean included or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, "comprising," "having," "including," or "containing" can also mean closed-ended, excluding additional, uncited elements or method steps.

[0063] In this invention, the term "fusion protein" refers to a novel protein produced by fusing a biologically active functional protein molecule with other natural proteins (fusion chaperones) using techniques such as genetic engineering. Fusion proteins can have two or more different functions. Furthermore, to facilitate purification and other processing, fusion proteins may further include protein tags and / or protease cleavage sites. The selection of protein tags and protease cleavage sites is known to those skilled in the art and is not particularly limited.

[0064] In this invention, the term "amino acid" may include natural amino acids, non-natural amino acids, amino acid analogs, and all their D and L stereoisomers.

[0065] In this invention, amino acid deletion may refer to the deletion of 1, 2 or 3 or more amino acids from the amino acid sequence, as long as the altered sequence completely or partially retains the activity of the original amino acid sequence.

[0066] In this invention, amino acid addition can refer to adding 1, 2 or 3 or more amino acids at any position at the C-terminus, N-terminus or between the C-terminus and N-terminus of the amino acid sequence, as long as the modified sequence completely or partially retains the activity of the original amino acid sequence.

[0067] In this invention, amino acid substitution refers to the replacement of an amino acid at a certain position in an amino acid sequence with another amino acid, as long as the altered sequence completely or partially retains the activity of the original amino acid sequence. Amino acid substitution can be conserved amino acid substitution, meaning that compared to the original amino acid sequence, several amino acids are replaced by amino acids with similar or related properties to form the sequence. For example, conserved substitution can be generated based on the following amino acid substitutions: Val, Leu, or Ile substitution for Ala; Lys, Gln, Asn, or His substitution for Arg; Gln, His, Lys, or Arg substitution for Asn; Glu or Asn substitution for Asp; Ser or Ala substitution for Cys; Asn or Glu substitution for Gln; Asp or Gln substitution for Glu; Ala substitution for Gly; Asn, Lys, Gln, or Arg substitution for His; Leu, Met, Ala, Val, or Phe substitution for... Substitution of Ile; substitution of Leu by Ile, Met, Ala, Val, or Phe; substitution of Lys by Asn, Gln, or Arg; substitution of Met by Ile, Leu, or Phe; substitution of Phe by Leu, Val, Ile, Ala, or Tyr; substitution of Pro by Ala; substitution of Ser by Thr; substitution of Thr by Ser or Val; substitution of Trp by Phe or Tyr; substitution of Tyr by Trp, Phe, Thr, or Ser; and substitution of Val by Phe, Ala, Met, Ile, or Leu. Amino acid substitutions can also be non-conserved amino acid substitutions.

[0068] In this invention, "flexible linkers" include, but are not limited to, hydrocarbon linkers and peptide linkers. Peptide linkers consist of straight-chain or branched amino acids linked by peptide bonds, such as peptide linkers containing small nonpolar (e.g., Gly) and / or polar (e.g., Ser or Thr) amino acid residues, specifically Gly, Gly-Gly, Ser-Gly-Gly, Ser-Gly-Gly-Gly, Gly-Gly-Ser-Gly-Gly, Gly-Ser-Ser-Gly-Gly, GSG, etc. Each linker (e.g., first, second, third linker) described herein may be independently selected from linkers of the above sequences, or from linkers of multiple sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12).

[0069] As used herein, the term "nucleic acid" refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from naturally occurring genes, modified to contain nucleic acid segments in a manner not originally present in nature, or is synthetic. The nucleic acid molecule may contain one or more control sequences. Nucleic acids can be codon-optimized nucleic acids, such as those codon-optimized for expression in E. coli cells.

[0070] The term "operably linked" refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide, such that the control sequence directs the expression of the coding sequence.

[0071] As used in this article, "DPEG" refers to discrete polyethylene glycol. DPEG4 indicates that the number of repeating glycol residues is 4.

[0072] The degree of association between two amino acid sequences or two nucleotide sequences is described by the parameter “sequence identity”. For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) implemented by the Needleman program in the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (preferably version 5.0.0 or later) is used to determine the sequence identity between two amino acid sequences. The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needleman labeled “longest identity” (obtained using the non-simplification option) is used as the identity percentage and calculated as follows:

[0073] (identical residues × 100) / (alignment length - total number of vacancies in the alignment)

[0074] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, ibid.) implemented by the Needleman program in the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, ibid.) (preferably version 5.0.0 or later) is used to determine sequence identity between two deoxynucleotide sequences. The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needleman labeled "Longest Identity" (obtained using the non-simplified option) is used as the identity percentage and calculated as follows:

[0075] (identical deoxyribonucleotides x 100) / (alignment length - total number of vacancies in the alignment)

[0076] In this invention, suitable vectors are known in the field of vector construction, including promoter selection and other regulatory elements, such as enhancer elements. The vectors described in this invention comprise sequences suitable for introduction into cells. For example, the vector can be an expression vector in which the coding sequence of a fusion protein is controlled by its own cis-regulatory elements, and the vector is designed to facilitate gene integration or gene replacement in host cells. Those skilled in the art will understand that in this invention, "vector" includes DNA molecules, such as plasmids, bacteriophages, viruses, or other vectors. It may contain one or more heterologous or recombinant nucleotide sequences. Suitable bacteriophage and viral vectors include, but are not limited to: λ-phage, EMBL-phage, simian virus, bovine wart virus, Epstein-Barr virus, adenovirus, herpesvirus, mouse sarcoma virus, murine mammary cancer virus, lentivirus, etc.

[0077] In this invention, the host cell can be a eukaryotic cell, such as fungi and yeast, or a prokaryotic cell, such as Enterobacteriaceae bacteria.

[0078] In this invention, any pharmaceutically acceptable excipients include, but are not limited to, diluents, osmotic pressure regulators, stabilizers, antibacterial agents, disintegrants, binders, fillers, lubricants, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, colorants, etc.

[0079] It should be understood that, in practical applications, the protein of the present invention or its pharmaceutical salt, its derivatives or their pharmaceutical salt, the above-mentioned conjugates, the above-mentioned polymers and the above-mentioned compositions can be given directly to patients as drugs, or given to patients after being mixed with suitable carriers or excipients.

[0080] In this invention, no limitation is made to the form of administration of the provided pharmaceutical compositions, including, for example, injection (intra-arterial, intravenous, intramuscular, intraperitoneal, subcutaneous), mucosal, oral (oral solid dosage form, oral liquid dosage form), rectal, inhalation, implantation, and topical (e.g., ocular) administration. Non-limiting examples of oral solid dosage forms include, but are not limited to, powders, capsules, lozenges, granules, tablets, etc. Non-limiting examples of oral or mucosal liquid dosage forms include, but are not limited to, suspensions, tinctures, elixirs, solutions, etc. Non-limiting examples of topical dosage forms include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Non-limiting examples of parenteral dosage forms include, but are not limited to, solutions for injection, dry powders for injection, suspensions for injection, emulsions for injection, etc. The compositions of this invention can also be formulated into controlled-release or delayed-release dosage forms (e.g., liposomes or microspheres).

[0081] The problem the invention aims to solve

[0082] The purpose of this invention is to provide a fusion protein that is an inactivating agent for SARS-CoV-2 and its mutant strains, as well as other coronaviruses that use ACE2 as a cell receptor. This protein inactivating agent can trigger a conformational change in the S2 subunit by binding to the receptor-binding domain (RBD) in the S1 subunit of the viral envelope protein via ACE2, thereby exposing the HR1 domain and causing the binding of coronavirus peptide therapeutics such as EK1 peptide that target the HR1 domain. Ultimately, through a "double-lock mechanism," the virus particle loses its infectivity.

[0083] Solution for solving the problem

[0084] A first aspect of the present invention provides a fusion protein of inactivated SARS-CoV-2 and its mutant strains, as well as other coronaviruses that use ACE2 as a cell receptor. Specifically, the present invention generates a novel bifunctional fusion protein by directly linking or linking a coronavirus peptide therapeutic agent targeting the HR1 domain, such as the ACE2 peptidase domain and the EK1 peptide, to the HR1 domain via a flexible linker.

[0085] The ACE2 peptidase domain can be at least 615 consecutive amino acids from the N-terminus of the human ACE2 protein. Coronavirus peptide therapeutics targeting the HR1 domain can be coronavirus peptide therapeutics such as the EK1 peptide, as previously claimed in this invention's patent application (publication number CN107022008A, invention title: Peptide for Broadly Inhibiting Human Coronavirus Infection and Its Application). The flexible linker can be a short peptide consisting of 5 amino acid residues. The C-terminus of the fusion protein may optionally contain a protein tag, which is linked to the last amino acid at the C-terminus of the protein via an enzyme cleavage site, such as the HRV 3C cleavage site. Preferably, a fusion protein of ACE2-linker-EK1 protein (i.e., AL2E, AL5E, AL6E, and AL7E) can be obtained. Particularly preferred is a fusion protein with the amino acid sequence shown in SEQ ID NO. 5, named AL5E. The only difference between AL2E, AL5E, AL6E and AL7E is the length of the connector, which represents (GGGGS)2, (GGGGS)5, (GGGGS)6 and (GGGGS)7 respectively.

[0086] The embodiments described herein do not involve the removal of the His tag; however, those skilled in the art should understand that the His tag and restriction enzyme sites have no inhibitory effect on the virus. The fusion protein (SEQ ID NO:7) after the removal of the His tag and restriction enzyme sites has the same antiviral effect as the fusion protein of SEQ ID NO:5.

[0087] The underlined portion in the above sequence represents the ACE2 peptidase domain of the AL5E protein, which consists of 615 consecutive amino acids at the N-terminus of the ACE2 protein. The specific sequence is shown in SEQ ID NO.1. The wavy underlined portion in the above sequence represents the EK1 polypeptide. The specific sequence is shown in SEQ ID NO.2. The bold portion in the above sequence represents the flexible linker in the AL5E protein.

[0088] This invention also provides that, in addition to the ACE2 peptidase domain and the EK1 polypeptide, the fusion protein may also contain a protein or other functional protein for extending half-life. The structure of this fusion protein can be shown as ACE2 peptidase domain - first linker - EK1 polypeptide - second linker - half-life extension portion. Those skilled in the art can routinely prepare such fusion proteins containing three functions. For example, the inventor's previously filed Chinese patent application 202310423264.7 describes a half-life extension portion, which discloses that the amino acid sequence of SEQ ID NO:12 (DCAWHLGELVWCT) can extend the half-life. Furthermore, the inventors previously filed a Chinese patent CN202180001804.1 describing derivatives of coronavirus polypeptide therapeutics, in which EK1-plam:SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL-GSG-DPEG4-K (palmit ic) and EK1-chol:SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL-GSGSG-DPEG4-C (cholesterol) were verified to have inhibitory activity against coronavirus pseudoviruses such as 2019-nCoV. The polypeptide derivative may contain SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL-(GSG)n-DPEG4-K-palmitic acid or a salt or ester thereof, or SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL-(GSGSG)n-DPEG4-C-cholesterol or a salt or ester thereof, where n is 1 or 2. Those skilled in the art can use such derivatives to replace the EK1 polypeptide used in the examples herein.

[0089] A second aspect of the invention provides a nucleic acid encoding the fusion protein of the invention, wherein the nucleotide sequence encoding the nucleic acid comprises the sequence shown in SEQ ID NO.3 or SEQ ID NO.4, or a derivative thereof according to artificial design and codon optimization.

[0090] SEQ ID NO.3 (ACE2 peptidase domain encoding nucleotide):

[0091]

[0092]

[0093] SEQ ID NO.4 (EK1 polypeptide encoding nucleotide):

[0094]

[0095] A third aspect of this invention provides a recombinant expression vector. The expression vectors selected in this invention can stably exist and autonomously replicate in various hosts, including prokaryotic or eukaryotic cells, such as conventional plasmids (pFuse-hIgG1-Fc2 series), shuttle vector PNV 18.1, phage vectors, or viral vectors. The nucleotide sequences of this invention are cloned into the vector through molecular biological operations such as enzyme digestion and ligation to construct the recombinant expression vector.

[0096] A fourth aspect of the present invention provides a recombinant host cell selected from prokaryotic cells, yeast, or eukaryotic cells, and further selected from Escherichia coli, Rhodococcus rubrum, Bacillus subtilis, and yeast. The recombinant expression vector can be transformed into the host cell to obtain the corresponding genetically engineered bacteria.

[0097] A fifth aspect of the present invention provides a pharmaceutical composition comprising a fusion protein, an encoding nucleic acid, a recombinant expression vector and / or a recombinant host cell, and optionally pharmaceutically acceptable excipients. The present invention does not limit the formulation of the pharmaceutical composition, which can be determined by those skilled in the art according to actual needs.

[0098] A sixth aspect of the invention provides the use of fusion proteins, encoding nucleic acids, recombinant expression vectors, recombinant host cells, and / or pharmaceutical compositions in the preparation of medicaments for treating infections caused by SARS-CoV-2 and its mutant strains, as well as other coronaviruses that use ACE2 as a cell receptor. The fusion proteins, encoding nucleic acids, recombinant expression vectors, recombinant host cells, and / or pharmaceutical compositions can be used for the prevention and / or treatment of infections caused by SARS-CoV-2 and its mutant strains, as well as other coronaviruses that use ACE2 as a cell receptor.

[0099] A seventh aspect of the present invention provides a method for preventing and / or treating SARS-CoV-2 and its mutant strains and other coronaviruses that use ACE2 as a cell receptor, comprising administering to a subject a preventive and / or therapeutically effective amount of a fusion protein, encoding a nucleic acid, a recombinant expression vector, a recombinant host cell, and / or a pharmaceutical composition.

[0100] fake virus preparation

[0101] In this paper, pseudoviruses can be prepared using methods known in the art. For example, pseudoviruses can be prepared by transfecting 293T cells with plasmids including the HIV backbone plasmid pNL4-3. Luc.R. - .E - Expression plasmids for the S protein of different SARS-CoV-2 mutant strains. The amino acid sequences of the S protein of different SARS-CoV-2 mutant strains are known. The table below provides expression plasmids for the S protein of various SARS-CoV-2 mutant strains.

[0102] Table 4:

[0103]

[0104]

[0105] Example

[0106] The present invention is further illustrated by the following embodiments, but any embodiment or combination thereof should not be construed as limiting the scope or implementation of the present invention. The scope of the present invention is defined by the appended claims, and those skilled in the art will clearly understand the scope defined by the claims in conjunction with this specification and common knowledge in the art. Without departing from the spirit and scope of the present invention, those skilled in the art can make any modifications or changes to the technical solutions of the present invention, and such modifications and changes are also included within the scope of the present invention.

[0107] Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments without a specified manufacturer are commercially available, conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. Those skilled in the art should understand that the invention can be practiced even without certain specific details. In other embodiments, methods, means, equipment, and steps well-known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0108] The protein materials used in the examples were designed by the inventors based on a novel mechanism by which secretory ACE2 protein, upon binding to the viral S1 protein RBD, induces a conformational change in S2 to expose HR1, facilitating EK1 peptide binding. The ACE2 peptidase domain and EK1 peptide were designed using linkers of different lengths to create ACE2-linker-EK1 proteins (i.e., AL2E, AL5E, AL6E, and AL7E). The proteins were then purified using a histidine octamer tag at the C-terminus, and the correctness and purity of the proteins were confirmed by gel filtration chromatography and Coomassie brilliant blue staining.

[0109] Example 1: Identification of AL2E, AL5E, AL6E and AL7E proteins by gel filtration chromatography and Coomassie brilliant blue staining

[0110] Expression plasmids encoding the AL2E, AL5E, AL6E, and AL7E genes (expression vector pFuse-hIgG1-Fc2) were synthesized and provided by Nanjing Genscript Biotech Co., Ltd. The expression plasmids encoding the AL2E, AL5E, AL6E, and AL7E genes were transfected into a 2*10n density vector using EZ-Trans transfection reagent (purchased from Liji Biotechnology, catalog number PD1311-01) according to the manufacturer's instructions. 6 Expi 293 suspension cells (purchased from Thermo Fisher) were collected at a density of cells / mL. Five days after transfection, the cell culture supernatant (containing the target protein) was collected by centrifugation and filtered through a 0.45 μm filter to remove cell debris. Ni-NTA beads (purchased from Tiandi Renhe, catalog number 191024) were added, and the mixture was incubated on a rotary mixer at 4°C for 10 hours. The liquid containing the beads was added to a protein purification gravity column (purchased from Beyotime), followed by washing with phosphate buffer containing 10 mM imidazole to remove contaminating proteins. Finally, the target protein was obtained by elution with phosphate buffer containing 250 mM imidazole. The harvested target protein was centrifuged and filtered through a 30 kDa protein ultrafiltration tube (purchased from Merck) to remove imidazole. The protein preservation solution was completely replaced with phosphate buffer (pH = 7.4), the concentration was determined, and the protein was aliquoted and stored at -80°C for subsequent experiments.

[0111] The AL2E nucleotide sequence is as follows (SEQ ID NO:8):

[0112]

[0113] The AL5E nucleotide sequence is as follows (SEQ ID NO:9):

[0114]

[0115]

[0116] The AL6E nucleotide sequence is as follows (SEQ ID NO:10):

[0117]

[0118]

[0119] The AL7E nucleotide sequence is as follows (SEQ ID NO:11):

[0120]

[0121]

[0122] AL2E, AL5E, AL6E, and AL7E proteins, purified by affinity chromatography, were further purified using a Superose 6 Increase 10 / 300GL gel filtration chromatography column (GE Healthcare), and the purified proteins were then exchanged for PBS buffer. Specifically, the Superose 6 Increase 10 / 300GL gel filtration chromatography column (GE Healthcare) was loaded into an AKTA Avant protein purification system (GE Healthcare), with a flow rate of 0.8 mL / min and a column pressure of 1.5 MPa. The column was washed with 48 mL of PBS buffer. Subsequently, 0.5 mL of AL2E, AL5E, AL6E, and AL7E proteins at a concentration of 5 mg / mL were injected into the loading loop through the injection module of the instrument. The program was then set to loading via the loading loop, with a flow rate of 0.8 mL / min, a column pressure of 1.5 MPa, an elution volume of 24 mL, and automatic collection upon reaching an absorbance of 280 nm. The program was then initiated. Similarly, the AL5E, AL6E, and AL7E proteins were processed according to the above procedure. This process automatically replaces the protein-containing solution with PBS buffer. SDS-PAGE experiments were then performed on the AL2E, AL5E, AL6E, and AL7E proteins. Specifically, 10 μL of 5×SDS-PAGE protein loading buffer (Yisheng Biotechnology, catalog number 20315ES20) was added to 40 μL of protein solution. The mixture was heated at 100℃ for 10 minutes, cooled in an ice bath, and then 20 μL of sample was added to the wells of the SDS-PAGE protein electrophoresis gel. The voltage was set to 80V, and electrophoresis was performed for 20 minutes. The voltage was then adjusted to 120V, and electrophoresis was performed for 40 minutes. After electrophoresis, the gel was removed, and the protein gel was stained with protein staining solution (BioSharp, catalog number 243183). The results were photographed and saved after staining.

[0123] Results: The elution peaks of the four proteins in gel filtration chromatography were uniform and symmetrical, indicating good homogeneity in solution. SDS-PAGE analysis showed that the apparent molecular weights of the fusion proteins AL2E, AL5E, AL6E, and AL7E were the same as their actual molecular weights, indicating correct expression of the fusion proteins. Furthermore, the purity reached over 99%, making them suitable for subsequent experiments. Gel filtration chromatography and SDS-PAGE results are shown below. Figure 1 As shown.

[0124] Example 2: Screening results of pseudovirus inhibition of SARS-CoV-2 VOC and Omicron mutant strains by AL2E, AL5E, AL6E, and AL7E proteins.

[0125] Preparation of pseudoviruses for SARS-CoV-2 VOC and Omicron mutant strains (refer to Xing L., et al. A five-helix-based SARS-CoV-2 fusion inhibitor targeting Heptad Repeat 2domain against SARS-CoV-2 and its variants of concern. Viruses. 2022 Mar 13; 14(3):597): Plasmids encoding the S protein of SARS-CoV-2 VOC and Omicron mutant strains (plasmid name pcDNA 3.1, synthesized by Beijing BGI Genomics Co., Ltd.) were co-transfected with pNL4-3.Luc.RE plasmid (containing luciferase expression gene, purchased from Addgene) at a mass ratio of 1:2 into HEK293T cells (purchased from ThermoFisher) 24 hours after passage. 10 hours after transfection, the original cell culture medium was discarded and replaced with fresh 10% serum DMEM (purchased from Gibco, catalog number C12430500BT). 48 hours after changing the medium, the cell culture supernatant was collected, and after centrifugation at 3000 rpm to remove cell debris, the corresponding pseudoviruses were obtained (as shown in Table 1), aliquoted and frozen at -80℃ for later use.

[0126] Human colon cancer cells (Caco-2) expressing the ACE2 receptor were divided into 10 groups. 4 Cells were seeded per well in 96-well plates and incubated at 37°C. AL2E, AL5E, AL6E, or AL7E proteins were serially diluted 5-fold with DMEM medium in the 96-well U-bottom plates, ranging from 100 nM to 0.0064 nM. Then, an equal titer and volume of SARS-CoV-2 VOC or Omicron mutant pseudovirus were added. The plates were incubated at 37°C for 1 hour. The original culture medium was discarded, and 100 μL of the fusion protein / pseudovirus mixture (drug-treated wells) or pseudovirus (virus wells) was added to the target cells after supernatant removal. Pseudovirus-free treatment groups were used as cell wells. After incubation at 37°C for 12 hours, the medium was replaced with fresh DMEM medium containing 10% serum. After 72 hours, discard the original culture medium in the 96-well cell culture plate. Add 40 μL of cell lysis buffer (Promega, catalog number E1531) to each well and lyse the cells on a decolorizing shaker for 45 minutes. Transfer 30 μL of cell lysis buffer from each well to a 96-well microplate, and then add 30 μL of firefly luciferase substrate (Promega, catalog number E1501) to each well. Place the microplate in a microplate reader (PerkinElmer) and read the fluorescence value of each well. Calculate the inhibition rate curve and the median effective dose (IC50) of the drug. 50Specifically, the virus inhibition rate of each well in the drug-treated wells was calculated using the following formula: Inhibition rate = (Fluorescence value of virus wells - Fluorescence value of drug-treated wells) / (Fluorescence value of virus wells - Fluorescence value of cell wells) * 100%. Virus wells do not include fusion protein treatment, drug-treated wells include fusion protein treatment, and cell wells do not include virus treatment. The calculated virus inhibition rate for each well was imported into Graphpad Prism software, and the Variable Slope was selected in Dose-Response-Inhibition mode to generate inhibition rate curves and IC50 values. 50 Numerical value.

[0127] The final virus inhibition curve and calculated IC 50 The values ​​are as follows: Figure 2 As shown in Table 1, the IC50 of the AL2E protein against various pseudoviruses... 50 The IC50 concentration of AL5E protein against various pseudoviruses ranges from 0.09 to 1.03 nM. 50 The IC50 of AL6E protein against various pseudoviruses ranges from 0.03 to 0.93 nM. 50 The IC50 of the AL7E protein against various pseudoviruses ranges from 0.07 to 1.96 nM. 50 The concentration ranged from 0.11 to 1.14 nM. Unexpectedly, these results indicate that among linker lengths, the AL5E protein exhibited the best inhibitory effect against various pseudoviruses, being 27 to 600 times more potent than the ACE2 protein alone. Furthermore, the IC50 of the AL5E protein against various pseudoviruses decreased with viral mutation. 50 The lack of significant differences indicates that the AL5E protein has broad-spectrum inhibitory activity against all variants.

[0128] Table 1: Inhibitory activity of AL2E, AL5E, AL6E, or AL7E proteins against pseudoviruses of SARS-CoV-2 VOC and Omicron mutant strains.

[0129]

[0130] Example 3: Inhibition of infection by AL5E protein against live Omicron mutant strain BA.2.2 subtype virus

[0131] African green monkey kidney cells (Vero-E6) were processed at a ratio of 1.6*102 4Inoculate 96-well plates with 100 PCR aliquots per well and incubate at 37°C for 12 hours. In the 96-well U-bottom plates, serially dilute AL5E protein (0.2 nM–500 nM), ACE2 protein (1.6 nM–5000 nM), and EK1 peptide (16 nM–50000 nM) with DMEM medium. Then add an equal volume of live SARS-CoV-2BA.2.2 virus (this experiment was conducted in the P3 laboratory of Shanghai Medical College, Fudan University; the virus was provided by the aforementioned institution) (100 TCID). 50 Incubate at 37°C for 1 hour. Discard the original culture medium in the cell plate, and add 100 μL of AL5E protein / virus mixture, ACE2 protein / virus mixture, or EK1 peptide / virus mixture (drug treatment wells) or live virus (virus wells) to the target cells Vero-E6 after removing the supernatant. The treatment group without virus is the cell well. After incubating at 37°C for 12 hours, replace with DMEM medium containing 1% methylcellulose and 2% FBS. After 72 hours, discard the culture medium, add 4% paraformaldehyde to fix the cells, and then add 0.2% Triton X-100 to disrupt the cell membrane and inactivate the virus. Then add blocking solution (blocking solution is PBS buffer with 3% bovine serum albumin) and block at 37°C for 2 hours. Next, add rabbit anti-SARS-CoV-2N protein antibody (purchased from Sinocare, catalog number 40588-T62) (1:1500 dilution) and incubate at 37°C for 2 hours. After washing three times with PBS, FITC-labeled goat anti-rabbit antibody (purchased from Thermo Fisher Scientific, catalog number A32731) (1:2000 dilution) was added and incubated at 37°C for 1 hour. After washing four times with PBS, the number of fluorescent cells in each well was measured using a CTL fluorescent spot counter. The virus inhibition rate in each well treated with the drug was calculated using the following formula: Inhibition rate = (Number of fluorescent cells in virus wells - Number of fluorescent cells in drug-treated wells) / (Number of fluorescent cells in virus wells - Number of fluorescent cells in cell wells) * 100%. Virus wells do not include fusion protein treatment, drug-treated wells include fusion protein treatment, and cell wells do not include virus treatment. The calculated virus inhibition rate for each well was imported into Graphpad Prism software, and the Variable Slope was selected in Dose-Response-Inhibition mode to generate inhibition rate curves and IC50 values. 50 Numerical values. Plot inhibition rate curves and calculate the half-maximal effective dose (IC50) of the drug. 50 The final viral inhibition curve and calculated IC50 were generated. 50 Values ​​such as Figure 3 As shown. The IC50 of the AL5E protein against live BA.2.2 virus was calculated. 50The concentration was 5.972 nM, and its inhibitory effect was better than that of ACE2 protein alone (IC50). 50 =262.5) 44 times stronger, which indicates that the AL5E protein has a good inhibitory effect on both pseudovirus and live virus cell infection models.

[0132] Example 4: Results of AL5E protein inactivation of SARS-CoV-2 VOC and Omicron mutant pseudovirus

[0133] You can refer to Wang X., et al. Synergistic effect by combining a gp120-binding protein and a gp41-binding antibody to inactivate HIV-1 virions and inhibit HIV-1 infection. Molecules. 2021 Mar 31; 26(7):1964 for experiments on AL5E protein inactivation of SARS-CoV-2 VOC and Omicron mutant pseudovirus.

[0134] Specifically, human colon cancer cells (Caco-2) expressing the ACE2 receptor were divided into 10... 4Cells were seeded per well in 96-well plates and incubated at 37°C for 24 hours. AL2E, AL5E, AL6E, or AL7E proteins were serially diluted 4-fold in DMEM medium in EP tubes, ranging from 500 nM to 0.488 nM. Equal titers and volumes of SARS-CoV-2 VOC and Omicron mutant pseudoviruses (preparation process as described in Example 2) were then added. The cells were incubated at 4°C for 1 hour. PEG-6000 to a final concentration of 10% was added to the protein / pseudovirus mixture or pseudovirus alone to precipitate the virus. The group without added virus served as the cell group. Subsequently, the cells were centrifuged at 12,000 rpm for 1 hour at 4°C, the supernatant was discarded, and 3% PEG-6000 (diluted with 10 mg / mL BSA) was added to wash away any remaining protein on the virus surface. After three washes, the virus was resuspended in DMEM and added to Caco-2 target cells after removing the supernatant. The cells were incubated at 37°C for 12 hours, then the medium was replaced with fresh DMEM containing 10% FBS. The luciferase assay was performed 72 hours later using the same method as in Example 2, calculating the virus inactivation rate per well using the following formula: Inactivation rate = (Number of fluorescent cells in virus wells - Number of fluorescent cells in drug-treated wells) / (Number of fluorescent cells in virus wells - Number of fluorescent cells in cell wells) * 100%. The calculated virus inhibition rate for each well was then imported into Graphpad Prism software. The Variable Slope setting was selected under Dose-Response-Inhibition mode to generate inhibition rate curves and EC50. 50 Numerical values. Calculate the inactivation rate curve and the median effective dose (EC50) of the drug. 50 ).

[0135] The final virus inactivation curve and calculated EC 50 The values ​​are as follows: Figure 4 As shown in Table 2. The AL5E protein's EC50 response to various pseudoviruses... 50 The concentration ranges from 1.29 to 8.72 nM, and its virus inactivation effect is 22 to 507 times that of the ACE2 protein alone. Furthermore, with viral mutation, the AL5E protein's EC50 activity against various pseudoviruses... 50 The lack of significant differences indicates that the protein has broad-spectrum inactivation activity against all variants.

[0136] Table 2. Inactivation activity of AL5E protein against pseudoviruses of SARS-CoV-2 VOC and Omicron mutant strains.

[0137]

[0138] Example 5: Inhibition and inactivation results of AL5E protein inhibition and inactivation of other coronaviruses using ACE2 as a cell receptor

[0139] Preparation of SARS-CoV, human coronavirus NL63, and bat-derived SARS-CoV-like coronaviruses: The S protein encoding gene plasmids (pcDNA 3.1) of SARS-CoV, human coronavirus NL63, and bat-derived SARS-CoV-like coronaviruses (WIV1 and Rs3367) (synthesized by Beijing Huada Liuhe Co., Ltd.) were co-transfected with pNL4-3.Luc.RE plasmid (containing a luciferase expression gene, purchased from Addgene) at a mass ratio of 1:2 into HEK293T cells (purchased from Thermo Fisher Scientific) 24 hours after passage. Ten hours after transfection, the original cell culture medium was discarded and replaced with fresh 10% serum DMEM (purchased from Gibco, catalog number C12430500BT). Forty-eight hours after medium replacement, the cell culture supernatant was collected, centrifuged at 3000 rpm to remove cell debris, and the corresponding pseudoviruses were obtained, aliquoted, and frozen at -80°C for later use.

[0140] (1) Results of AL5E protein inhibiting other coronaviruses that use ACE2 as a cell receptor

[0141] Human colon cancer cells (Caco-2) expressing the ACE2 receptor were divided into 10 groups. 4 Inoculate 1 sample per well into a 96-well plate and incubate at 37°C for 24 hours. In the 96-well U-type plates, serially dilute AL2E, AL5E, AL6E, or AL7E proteins 5-fold to varying concentrations (100 nM to 0.0064 nM). Then add equal titers and volumes of SARS-CoV, human coronavirus NL63, or bat-derived SARS-CoV-like coronavirus. The remaining procedures are the same as described in Example 2.

[0142] The final virus inhibition curve and calculated IC 50 Values ​​such as Figure 5 (a) and Table 3 are shown. The IC50 of the AL5E protein against four pseudoviruses was calculated. 50 The concentration ranged from 0.69 to 5.90 nM, and its inhibitory effect was 14 to 70 times stronger than that of ACE2 protein alone. This result indicates that AL5E protein also has a good inhibitory effect on other coronaviruses that use ACE2 as a cell receptor.

[0143] Table 3. Inhibitory and inactivating activities of AL5E protein against other coronaviruses that use ACE2 as a cell receptor.

[0144]

[0145] (2) Results of AL5E protein inactivation of other coronaviruses using ACE2 as a cell receptor

[0146] Human colon cancer cells (Caco-2) expressing the ACE2 receptor were divided into 10 groups. 4 Inoculate 96-well plates with 1 sample per well and incubate at 37°C for 24 hours. AL2E, AL5E, AL6E, or AL7E proteins are serially diluted 4-fold in EP tubes to a concentration range of 250 nM to 0.244 nM. Then, add equal titers and volumes of pseudoviruses of SARS-CoV, human coronavirus NL63, and bat-derived SARS-CoV-like coronaviruses (WIV1 and Rs3367) (prepared as described in Example 5(1)). The remaining procedures are the same as described in Example 2.

[0147] The final virus inactivation curve and calculated IC 50 Values ​​such as Figure 5 (b) and Table 3 are shown. Calculations were performed to determine the EC5E protein response of the AL5E protein to four pseudoviruses. 50 The concentrations ranged from 1.08 to 18.13 nM, and the inhibitory effect was 26 to 540 times stronger than that of ACE2 protein alone. These results indicate that the AL5E protein also maintained good inactivation efficacy against other coronaviruses that use ACE2 as a cellular receptor. Importantly, compared to ACE2 protein alone, the AL5E protein showed a greater improvement in inactivation efficacy than inhibition against these four pseudoviruses, suggesting that this protein may have a better effect on inactivating free viruses in these coronaviruses that have previously broken out, may recur, or pose a potential threat to humans.

[0148] Example 6: Results of AL5E protein prevention and protection of K18-ACE2 transgenic mice from human coronavirus NL63 infection.

[0149] To determine the efficacy of AL5E protein in preventing and protecting K18-ACE2 transgenic mice from human coronavirus NL63 (a gift from the Institute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention), 6-8 week old female human ACE2 transgenic mice were purchased from Jicui Pharmaceutical Co., Ltd. for viral challenge. Six hours before challenge (7000 PFU), mice treated with AL5E protein prepared in Example 1 were administered via pulmonary nebulization at a dose of 1.5 mg / kg body weight as the prevention group. At 12, 24, 48, 72, and 96 hours after challenge, mice treated with AL5E protein were administered via pulmonary nebulization at a dose of 15 mg / kg body weight as the treatment group. Five days after challenge, the mice were euthanized, and their lungs were removed and photographed to observe organ lesions. Subsequently, the lungs were fixed with 4% paraformaldehyde for histological examination (using HE staining, this experiment was commissioned to Wuhan Sewell Co., Ltd.). Simultaneously, RNA was extracted from the lung tissue after grinding, and the viral N gene copy number in the lungs was detected by RT-qPCR. Specifically, RNA was extracted using the RNAsimple Total RNA Kit (TIANGEN, catalog number DP419) following the manufacturer's instructions. The extracted RNA was then subjected to quantitative real-time PCR using the One-Step PrimeScrip RT-PCR Kit (Takara, catalog number R064A) to detect the copy number of the N gene in the lungs. The amplification primers were: SARS-CoV-2N-F: 5′-GGGGAACTTCTCCTGCTAGAAT-3′; SARS-CoV-2N-R: 5′-CAGACATTTTGCTCTCAAGCTG-3′, and the amplification probe was: SARS-CoV-2-N-probe: 5′-FAM-TTGCTGCTGCTTGACAGATT-TAMRA-3′. All primers and probes were synthesized by Beijing Liuhe Huada Co., Ltd. The amplification program was 45℃ for 5 minutes, 95℃ for 10 seconds, 95℃ for 10 seconds, 50℃ for 30 seconds, 72℃ for 30 seconds, and the cycle of steps 3 to 5 was repeated 30 times.

[0150] Statistical analysis was performed using GraphPad Prism 8.0. One-way ANOVA was used for comparisons among multiple groups, followed by Tukey's post-hoc test for multiple comparisons. P < 0.05 was considered statistically significant, and P < 0.001 was considered highly statistically significant. The results indicate that, as Figure 6As shown in (a), (b), and (c), the viral copy number in the lungs of mice in the prevention and treatment groups was significantly lower than that in the PBS control group. The lungs of mice in the prevention and control groups also showed fewer congestion and hemorrhagic lesions. HE staining also revealed less inflammatory cell infiltration, alveolar wall thickening, and alveolar hemorrhage. This further demonstrates that the AL5E protein can effectively prevent and protect mice from NL63 coronavirus infection and can reduce inflammation in the lungs.

[0151] Example 7: Results of AL5E protein inactivating human coronavirus NL63 and protecting K18-ACE2 transgenic mice from infection.

[0152] To determine the effectiveness of AL5E protein in inactivating human coronavirus NL63 (provided by the Institute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention) in protecting K18-ACE2 transgenic mice from infection, 7000 PFU of coronavirus NL63 was mixed with an equal volume of AL5E protein (prepared in Example 1) (15 mg / kg) and administered intranasally to mice. Remdesivir (purchased from MCE, catalog number 1809249-37-3) (25 mg / kg), a nucleoside analog inhibitor with no virus inactivation but with virus inhibition, was used as a negative control for virus inactivation, and an equal volume of PBS was used as a solvent control. On day 5 post-infection, mice were euthanized and processed according to the method described in Example 6.

[0153] The results show that, Figure 7 As shown in (a), (b), and (c), the viral copy number in the lungs of mice in the AL5E protein inactivation group was significantly lower than that in the PBS control group and the remdesivir treatment group, while there was no difference between the remdesivir treatment group and the PBS control group. Furthermore, the lungs of mice in the AL5E protein inactivation group showed less congestion and hemorrhage. Further histological examination revealed less inflammatory cell infiltration, alveolar wall thickening, alveolar hemorrhage, and lung cell necrosis and disintegration in the lungs of mice in the AL5E protein inactivation group.

[0154] The above results further demonstrate the advantage of using viral inactivation proteins over viral inhibitors in the early stages of viral infection, namely, that they can inactivate the virus in the very early stages when the virus is free and has not yet bound to cells, thus better protecting the body's tissues.

[0155] discuss

[0156] In this invention, the inventors first identified the correctness and purity of ACE2-EK1 proteins with different length linkers expressed in eukaryotes using gel filtration chromatography and Coomassie brilliant blue staining. Secondly, the inventors tested the inhibitory activity of AL2E, AL5E, AL6E, and AL7E proteins against pseudovirus infection using a newly established SARS-CoV-2 pseudovirus packaging and infection inhibition system. Previously, the inventors constructed pseudoviruses of a series of SARS-CoV-2 mutant strains by co-transfecting 293T cells with the viral S protein expression plasmid and the backbone plasmid pNL4-3. Based on this system, they successfully designed and evaluated a series of SARS-CoV-2 protein entry inhibitors with good activity (detailed methods can be found in Xing L., et al. A five-helix-based SARS-CoV-2 fusion inhibitor targeting Heptad Repeat 2domain against SARS-CoV-2 and its variants of concern. Viruses. 2022 Mar 13; 14(3):597). In this invention, the inventors used the same method to test the inhibitory activity of the invented AL2E, AL5E, AL6E, and AL7E proteins against SARS-CoV-2 VOC and Omicron mutant subtype pseudovirus infection. The results showed that, compared to the ACE2 peptidase-binding domain alone, the AL5E protein, among the fusion proteins with different linkers, exhibited the best activity, increasing by 27 to 600 times.

[0157] To further explore the effectiveness of the AL5E protein in this invention in inhibiting the original SARS-CoV-2 strain, as well as the VOC strain and the novel Omicron mutant subtype published by the WHO, this invention tested the inhibitory activity of the AL5E protein against the original SARS-CoV-2 strain, five VOC strains, and the novel Omicron mutant subtype (BA.1–BA.5, BF.7, XBB, BQ.1). The results showed that the AL5E protein had an IC50 value against different types of pseudoviruses. 50 The concentration range is 0.03–0.93 nM, which is considered a low nanomolar level. More importantly, its inhibitory effect is 27–600 times higher than that of ACE2 protein alone. Although pseudoviruses containing SARS-CoV-2 envelope proteins can effectively mimic the receptor binding and membrane fusion process of live virus infection, the reliable inhibitory activity of the drug still requires further testing with real live viruses. Therefore, this invention, based on the P3 laboratory of Fudan University, tested the inhibitory activity of AL5E protein against BA.2.2 live virus. The results showed that the IC50 of AL5E protein against BA.2.2 live virus was [missing information]. 50 Below 5 nM, it is basically consistent with the inhibitory activity against pseudovirus infection.

[0158] The fusion protein (especially the AL5E protein) in this invention not only exhibits good inhibitory activity against the original SARS-CoV-2 strain, VOC strains, and novel subtypes of the Omicron mutant strain, as well as live and pseudoviruses, but more importantly, it can directly act on free viruses, rendering them inactive. This is something that current small molecule inhibitors and other peptide inhibitors that act on viral proteases do not possess. The inventors used a protein-based virus inactivating agent to detect the virus inactivation effect of the AL5E protein against the original SARS-CoV-2 strain, five VOC strains, and novel subtypes of the Omicron mutant strain (BA.1–BA.5, BF.7, XBB, BQ.1). Previously, the inventors successfully designed and evaluated a series of HIV protein-based virus inactivators with good activity by co-incubating the virus with a protein inactivating agent, precipitating virus particles with PEG-6000, and washing to remove residual proteins (for detailed methods, refer to Wang X., et al. Synergistic effect by combining a gp120-binding protein and a gp41-binding antibody to inactivate HIV-1 viruses and inhibit HIV-1 infection. Molecules. 2021 Mar 31; 26(7):1964). In this invention, the inventors used the same method to test the inactivation activity of the invented AL5E protein against the original SARS-CoV-2 strain, five VOC strains, and a new subtype of the Omicron mutant strain (BA.1~BA.5, BF.7, XBB, BQ.1). The results showed that the AL5E protein was effective against different types of pseudoviruses. 50 The concentration ranges from 1.29 to 8.72 nM, which is considered low nanomolar levels. More importantly, its inactivation efficiency is 22 to 507 times higher than that of ACE2 protein alone. This indicates that the AL5E protein can efficiently inactivate both the original and mutant strains of SARS-CoV-2, and its inactivation effect is significantly better than that of ACE2 protein alone.

[0159] Among the coronaviruses that have already posed or have the potential to pose a threat to humans, SARS-CoV, human coronavirus NL63, and bat-derived SARS-like coronaviruses (WIV1, Rs3367), besides SARS-CoV-2, all use ACE2 as an entry receptor. Therefore, in this invention, the effectiveness of the AL5E protein in inhibiting and inactivating these viral strains was further investigated. The results showed that the IC50 of the AL5E protein for inhibiting these viruses was [not specified in the original text]. 50At 0.69–5.90 nM, its effect is 14–70 times greater than that of ACE2 protein alone. The AL5E protein's EC50 activity against the aforementioned viruses… 50 At 1.08–18.13 nM, its effect is 26–214 times that of ACE2 protein alone.

[0160] This invention further evaluated the preventive and protective effects of AL5E protein in a mouse model infected with human coronavirus NL63. The results showed that, compared to the PBS control group, administration of the drug 6 hours before challenge (1.5 mg / kg) and at 12, 24, 48, 72, and 96 hours after challenge (15 mg / kg) significantly reduced viral copy number and lung pathological damage in mice. Simultaneously, this invention evaluated the infection effect of a single dose of AL5E protein-inactivated virus in a mouse model infected with human coronavirus NL63. The results showed that the viral copy number in the mouse lungs after AL5E protein inactivation was significantly lower than that in the PBS control group and the group treated with remdesivir, an RNA-dependent RNA polymerase (RdRp) inhibitor that could not inactivate the virus.

[0161] In summary, the fusion protein presented in this paper (especially the AL5E protein) is a novel, broad-spectrum viral inhibitor and inactivator that targets both the viral RBD and HR1 domains through two functional domains, simultaneously inhibiting viral infection and inactivating free virus. It is effective against all coronaviruses that use ACE2 as a cell receptor. Its innovation lies in the highly efficient viral inactivation effect achieved by conjugating the ACE2 peptidase domain to the EK1 peptide, enhancing the activity of the ACE2 protein while simultaneously imparting viral inactivation to the EK1 peptide. Furthermore, its antiviral target is a novel discovery, previously unreported. This protein inactivator also exhibits good in vivo safety and demonstrates promising drug development potential.

Claims

1. A fusion protein comprising an ACE2 peptidase domain and a coronavirus peptide therapeutic agent targeting an HR1 domain, connected by a flexible linker, wherein the amino acid sequence of the ACE2 peptidase domain is SEQ ID NO: 1, and the amino acid sequence of the coronavirus peptide therapeutic agent is SEQ ID NO: 2 or 12, wherein the ACE2 peptidase domain is at the N-terminus of the fusion protein and the coronavirus peptide therapeutic agent is at the C-terminus of the fusion protein.

2. The fusion protein according to claim 1, wherein the flexible linker is (G). n1 (SGG) n2 (GGSGG) n4 (GSSGG) n5 Or (GGGGS) n6 Where n1 = 10 - 35; n2 = 3 - 13; n4, n5 or n6 = 2 - 10.

3. The fusion protein according to claim 2, wherein n4, n5 or n6 = 5.

4. The fusion protein according to claim 1, wherein the amino acid sequence of the fusion protein is SEQ ID NO:

7.

5. A fusion protein, which consists of the following (1) and (2): (1) A coronavirus peptide therapeutic agent that sequentially connects the ACE2 peptidase domain and the HR1 domain via a flexible linker from the N-terminus to the C-terminus, and (2) The enzyme cleavage site and / or purification tag at the C-terminus of (1), wherein the amino acid sequence of the ACE2 peptidase domain is SEQ ID NO: 1, and the amino acid sequence of the coronavirus polypeptide therapeutic agent is SEQ ID NO: 2 or 12.

6. The fusion protein of claim 5, wherein the flexible linker is (G). n1 (SGG) n2 (GGSGG) n4 (GSSGG) n5 Or (GGGGS) n6 Where n1 = 10 - 35; n2 = 3 - 13; n4, n5 or n6 = 2 - 10.

7. The fusion protein according to claim 6, wherein n4, n5 or n6 = 5.

8. The fusion protein according to any one of claims 5-7, wherein the restriction site is the HRV 3C restriction site and the purification tag is a His tag.

9. The fusion protein according to claim 8, wherein the His tag is an 8xHis tag.

10. The fusion protein according to claim 5, wherein the amino acid sequence of the fusion protein is SEQ ID NO:

5.

11. A nucleic acid encoding a fusion protein according to any one of claims 1-10.

12. The nucleic acid of claim 11, wherein the nucleic acid comprises a codon-optimized nucleotide sequence.

13. The nucleic acid of claim 12, wherein the nucleotide sequence is a codon-optimized nucleotide sequence for expression in eukaryotic or prokaryotic cells.

14. The nucleic acid of claim 13, wherein the nucleotide sequence is a codon-optimized nucleotide sequence for expression in yeast or Escherichia coli.

15. The nucleic acid according to claim 11, comprising the nucleotide sequence described in SEQ ID NO: 3 and / or 4.

16. The nucleic acid according to claim 11, wherein the nucleic acid comprises a nucleotide sequence selected from SEQ ID NO: 8-11.

17. A vector comprising a nucleic acid according to any one of claims 11-16.

18. The vector of claim 17, wherein the vector comprises an expression control element operatively linked to the nucleic acid, a polynucleotide of a purification tag, and / or a polynucleotide of a leader sequence.

19. The carrier according to claim 18, wherein the expression control element is selected from promoters, terminators, or enhancers.

20. The vector according to claim 18, wherein the purification tag is selected from His tag, GST tag, MBP tag, SUMO tag or NusA tag.

21. The vector according to any one of claims 17-20, wherein the vector is an expression vector, a cloning vector, a shuttle vector, or a viral vector.

22. The carrier according to any one of claims 17-20, wherein the carrier is a plasmid or a viscous particle.

23. The vector according to any one of claims 17-20, wherein the vector is a pFuse-hIgG1-Fc2 series plasmid or a pET series plasmid.

24. A host cell comprising a nucleic acid according to any one of claims 11-16 or a vector according to any one of claims 17-23.

25. The host cell of claim 24, wherein the host cell is a eukaryotic cell or a prokaryotic cell.

26. The host cell according to claim 24 or 25, wherein the eukaryotic cell is a yeast cell, an animal cell, and / or an insect cell, and / or the prokaryotic cell is an Escherichia coli cell.

27. The host cell according to claim 26 is Escherichia coli, Rhodococcus rubrum, Bacillus subtilis, or yeast.

28. A composition comprising one or more of the fusion protein according to any one of claims 1-10, the nucleic acid according to any one of claims 11-16, and the vector according to any one of claims 17-23.

29. The composition according to claim 28, wherein the composition is a pharmaceutical composition or a kit.

30. The composition of claim 28, wherein the composition comprises a pharmaceutically acceptable carrier.

31. The composition of claim 28, wherein the composition comprises a pharmaceutically acceptable diluent.

32. The composition according to claim 28, wherein the composition is a solid, liquid, or gel composition.

33. The composition according to claim 28, wherein the composition is an injectable composition or an oral composition.

34. The composition according to claim 28, wherein the composition is in the form of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, or suppositories.

35. The composition according to claim 34, wherein the pills are drop pills and the powder is lyophilized powder.

36. The composition of claim 28, wherein the composition further comprises another therapeutic agent against coronavirus.

37. The composition of claim 36, wherein the therapeutic agent is selected from Paxlovid, abavirin / romisvir, COVID-19 human immunoglobulin, convalescent plasma, glucocorticoids, and interleukin-6 inhibitors.

38. A method for preparing the fusion protein of any one of claims 1-10, comprising: (1) Culture the host cells according to any one of claims 24-27 under suitable culture conditions; (2) Harvesting host cells and / or culture medium containing the fusion protein; and (3) Purify the fusion protein.

39. The method of claim 38, wherein step (3) comprises, when the fusion protein contains a purification tag, using column separation with a ligand for the purification tag to separate the fusion protein and / or using a tool enzyme to cleave and remove the purification tag.

40. The method of claim 39, wherein the purification tag is a histidine tag.

41. The method of claim 40, wherein the purification tag is an 8xHis tag.

42. The method of claim 38, wherein the column separation is nickel column separation.

43. The method of claim 39, wherein when the fusion protein contains an HRV 3C cleavage site, the tool enzyme is an HRV 3C enzyme.

44. Use of a fusion protein according to any one of claims 1-10, a nucleic acid according to any one of claims 11-16, a vector according to any one of claims 17-23, a host cell according to any one of claims 24-27, and / or a composition according to any one of claims 28-37 in the preparation of a medicament or kit for treating or preventing coronavirus infection in a subject, the coronavirus containing ACE2 as a cell receptor.

45. The use according to claim 44, wherein the coronavirus is one or more of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), HCoV, RsSHC014-CoV or RsW1V1-CoV.

46. ​​The use according to claim 45, wherein HCoV is selected from HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1 strains; and SARS-CoV-2 is selected from the prototype, Alpha, Beta, Gamma, Delta, Lambda, BA.1, BA.2, BA.2.2, BA.2.9, BA.2.12.1, BA.2.75, BA.3, BA.4.6, BA.5, BF.7, XBB or BQ.1 viral strains.

47. The use according to claim 44, wherein the medicament is in the form of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, or suppositories.

48. The use according to claim 47, wherein the pills are drop pills and the powder is lyophilized powder.

49. The use according to claim 44, wherein the subject is a human or a mammal.

50. The use according to claim 49, wherein the mammal is a companion animal, a zoo animal, or a domestic animal.

51. A method for treating coronavirus infection in vitro for non-therapeutic purposes, comprising contacting coronavirus-infected cells in vitro with a fusion protein according to any one of claims 1-10, said coronavirus containing ACE2 as a cell receptor.

52. The method of claim 51, wherein the coronavirus is one or more of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), HCoV, RsSHC014-CoV or RsW1V1-CoV.

53. The method according to claim 52, wherein HCoV is selected from HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1 strains; and SARS-CoV-2 is selected from the prototype, Alpha, Beta, Gamma, Delta, Lambda, BA.1, BA.2, BA.2.2, BA.2.9, BA.2.12.1, BA.2.75, BA.3, BA.4.6, BA.5, BF.7, XBB or BQ.1 viral strains.

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