Trefoil factor 2 / interferon alpha 2 fusion protein and its use in the prevention and treatment of viral infections

Abstract

The present disclosure provides trefoil factor 2 / interferon α2 fusion proteins and their use in the prevention and treatment of viral infectious diseases. Specifically, the present disclosure provides fusion proteins that contain a trefoil factor 2 (TFF2) element that includes a TFF2 peptide, and an interferon α2 (IFNα2) element that includes an IFNα2 peptide fused to the TFF2 element. The present disclosure also provides uses of such fusion proteins in the prevention and / or treatment of viral infectious diseases, such as acute respiratory / enteric viral infections.

Description

[Technical field]

[0001] The present application relates to the biomedical field, specifically to a fusion protein of trefoil factor 2 (TFF2) and interferon alpha 2 (IFNα2), and its preparation and use in the treatment and prevention of viral infection diseases. [Background technology]

[0002] Viral infections pose great threats to the lives and health of humans and mammals, among which respiratory and enteric viral infections are the most common acute viral infections.

[0003] Acute respiratory viral infections are prone to cause epidemics, which severely threaten the life and health of human beings and have a negative impact on human life and socio-economic development, and there is a strong need to develop countermeasures to contain such epidemics. On the other hand, enteric viruses are sense single-stranded RNA viruses associated with human and mammalian diseases and are transmitted via the intestinal tract. Enteric viruses affect millions of people worldwide every year and are usually present in the respiratory secretions (e.g., saliva, sputum, or nasal mucus) and feces of infected individuals. Infections cause many symptoms, including mild respiratory illness (the common cold), hand, foot, and mouth disease, acute hemorrhagic conjunctivitis, aseptic meningitis, myocarditis, severe neonatal sepsis-like disease, acute flaccid paralysis, and associated acute flaccid myelitis.

[0004] Respiratory and enteric viruses are both mucosal infection viruses. Although the characteristics and infection methods of different viruses are not completely consistent, they share a common pathogenic mechanism: on the one hand, direct viral infection causes apoptosis and necrosis of target cells, damaging the structure and function of normal mucosa; on the other hand, viruses control the host's immune response, inhibiting the production of type I interferon and its signaling pathway, inducing a large amount of secretion of inflammatory cytokines and chemokines, generating a cytokine storm, recruiting a large number of immune cells to infiltrate mucosal tissues, severely destroying the mucosal structure, causing mucosal damage, inflammatory exudation, and ultimately causing the host to become ill or die. Therefore, for the common pathogenic mechanism of respiratory and enteric virus infection, it is necessary to limit viral replication while suppressing cytokine storm, promoting the repair of mucosal damage, and achieving the purpose of improving patient prognosis.

[0005] Currently, the most common anti-inflammatory drugs for the inflammatory response caused by viral infections are glucocorticoids, a type of steroid hormone. Glucocorticoids are part of the feedback mechanism of the immune system and can reduce certain aspects of immune function, which can effectively suppress inflammation. However, their side effects are also clear. Currently used glucocorticoid drugs act nonselectively and impair many healthy synthetic metabolic processes. Side effects of long-term administration of these drugs include iatrogenic hyperadrenocorticism, induction or aggravation of infection or metastasis of latent infection foci in the body, induction of peptic ulcers, induction of pancreatitis and fatty liver, iatrogenic adrenal insufficiency, induction of schizophrenia and epilepsy, and femoral head necrosis.

[0006] Interferon, either alone or in combination with other therapies, has been widely used as an antiviral drug in various viral infections and has an inhibitory effect against many currently known viruses. IFNα2, a typical antiviral drug, has been widely used in clinical practice, and its α-2b aerosol has a remarkable effect in the treatment of acute upper respiratory tract infections in children, and can effectively improve the clinical symptoms and vital signs of patients (Chen Qing et al., Analysis of the therapeutic effect of recombinant human interferon α-2b aerosol in children with acute upper respiratory tract infections, Biomedical Engineering and Clinical, 2019,23(04)); α-2b aerosol is a low-dose 20 When used in combination with oseltamivir at 100 μg to treat influenza A, it reduces the cellular inflammatory factor levels, increases the viral negative conversion rate, promotes symptom improvement, and is safe (Xu Guangfeng, Evaluation of the Effect of Combining Recombinant Human Interferon α-2b Aerosol and Oseltamivir in the Treatment of Influenza A, Journal of Shanxi College of Health and Medical Care, 2020,30(04). Animal experiments and clinical studies have shown that the combination of IFNα2 and ribavirin can effectively inhibit the replication of MERS-CoV and improve the symptoms and clinical outcomes of MERS. Both the 2003 SARS treatment plan and the latest version of the "Novel Coronavirus Infection Treatment Plan (Trial Eighth Edition)" recommend IFNα2 treatment (for adults, 5 million U or equivalent each time, 2 mL of sterile water for injection, atomized inhalation twice daily, treatment course of 10 days or less). In 2020, Pandit conducted a phase II study of polyethylene glycol interferon alfa-2b (PEG IFN-α2b) in moderate COVID-19 to evaluate its therapeutic efficacy and safety. Compared with standard treatment, subcutaneous administration of a single dose of 1 μg / kg PEG IFN-α2b plus standard treatment can reduce the duration of viral clearance and significantly improve clinical outcomes (Anuja Pandit, Efficacy and safety of pegylated interferon alfa-2b in moderate COVID-19: A phase II, randomized, controlled, open-label study, Int J Infect Dis. 202104;105:516-521).

[0007] TFF2, a small polypeptide secreted from the gastrointestinal tract, is involved in mucosal repair and is overexpressed during the inflammatory process. Addition of TFF2 has an inflammation-reducing effect and helps create the microenvironment necessary for tissue repair and promote tissue repair (Abdelaziz Ghanemi et al., Trefoil factor family member 2 (TFF2) as an inflammatory-induced and anti-inflammatory tissue repair factor,Animals (Basel). 2020 Sep 14;10(9):1646.). Previous studies have found that TFF2, a host-secreted peptide, can reduce pathological damage, promote the repair of lung tissue damage, exert protective effects, and improve the prognosis of influenza virus infection by suppressing inflammatory responses (CN105582526B). In 2019, a clinical study of COVID-19 pneumonia was urgently conducted to treat patients with moderate COVID-19 pneumonia using a combination of TFF2 and type I interferon kappa (IFN-k) by atomization inhalation. The results showed that this combination treatment could significantly shorten the time to nucleic acid negative conversion in COVID-19 patients, increase the proportion of patients with nucleic acid negative conversion, accelerate the improvement of CT scans, and shorten the hospitalization time of patients. At the same time, the levels of inflammatory cytokines in the plasma of treated patients were rapidly reduced (EClinicalMedicine 2020:100478 / 100547; China Patent Application No.: 202010239633.3). Summary of the Invention [Problem to be solved by the invention]

[0008] In the long-term battle between humanity and viral infections, there is a strong demand for the development of safer and more effective drugs and methods for preventing and treating viral infections. [Means for solving the problem]

[0009] The present disclosure provides effective active substances for preventing and / or treating viral infections more safely and effectively, their use in the preparation of medicaments, and methods for preventing and treating diseases.

[0010] In one aspect of the disclosure, a fusion protein is provided, which comprises one or more fusion units, each fusion unit comprising: (a) a trefoil factor 2 (TFF2) element, the TFF2 element comprising a TFF2 peptide or an active fragment thereof; (b) an interferon alpha 2 (IFNα2) element, the IFNα2 element comprising an IFNα2 peptide or an active fragment thereof; However, the TFF2 element and the IFNα2 element are fused at a molecular ratio of 1:1, and in each fusion unit, the TFF2 element is located at the N-terminus of the IFNα2 element.

[0011] In some embodiments, the TFF2 peptide or active fragment thereof is derived from a human, a primate, a rodent (eg, mouse, rat, guinea pig, hamster), dog, or cat.

[0012] In some embodiments, the TFF2 peptide or active fragment thereof comprises an amino acid sequence selected from the following: SEQ ID NO:8, SEQ ID NO:12, or an active fragment thereof (e.g., an amino acid sequence having at least 80% sequence identity to SEQ ID NO:8 or SEQ ID NO:12 and having TFF2 activity).

[0013] In some embodiments, the TFF2 peptide or active fragment thereof is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the following: SEQ ID NO:7, SEQ ID NO:11, or an active fragment thereof (e.g., a nucleic acid molecule having at least 80% sequence identity to SEQ ID NO:7 or SEQ ID NO:11 and capable of encoding an active TFF2 peptide).

[0014] In some embodiments, the IFNα2 peptide or active fragment thereof is derived from a human, a primate, a rodent (eg, mouse, rat, guinea pig, hamster), dog, or cat.

[0015] In some embodiments, the IFNα2 peptide or active fragment thereof comprises an amino acid sequence selected from the following: SEQ ID NO:10, SEQ ID NO:14, or an active fragment thereof (e.g., an amino acid sequence having at least 80% sequence identity to SEQ ID NO:10 or SEQ ID NO:14 and having IFNα2 activity).

[0016] In some embodiments, the IFNα2 peptide or active fragment thereof is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the following: SEQ ID NO:9, SEQ ID NO:13, or an active fragment thereof (e.g., a nucleic acid molecule having at least 80% sequence identity to SEQ ID NO:9 or SEQ ID NO:13 and capable of encoding an active IFNα2 peptide).

[0017] In some embodiments, the fusion protein further comprises a linker connecting the TFF2 element and the IFNα2 element and / or forming a peptide segment within the elements.

[0018] In some embodiments, the linker is a flexible linker comprising n amino acid residues, where n is an integer from 2-300.

[0019] In some embodiments, the linker is a glycine or a glycine / serine linker. n , (GS) n , (GGS) n , (GGGS) n , (GGGGS) n or (GGGGGS) n wherein n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0020] In some embodiments, the fusion protein comprises one or more consecutive or spaced TFF2 peptides and / or one or more consecutive or spaced IFNα2 peptides.

[0021] In some embodiments, the fusion protein further comprises an Fc region, wherein the Fc region does not contain a mutation. In some embodiments, the fusion protein comprises one or more mutations that reduce antibody-mediated ADCC and CDC activity. In some embodiments, the fusion protein comprises the amino acid mutations D265A and N297G according to the EU numbering system.

[0022] In some embodiments, the fusion protein further comprises a signal peptide, hi some embodiments, the signal peptide of the fusion protein is selected from a tPA2 signal peptide, a TFF2 signal peptide, an IL-2 signal peptide, a bPRL signal peptide, and a CD33 signal peptide.

[0023] In some embodiments, the fusion protein further comprises a marker, e.g., a marker for use in purification, detection, or localization, e.g., a fluorescent marker, a non-radioactive nuclide marker, a biotin-based marker, a phosphorylation modification mark, or a peptide tag.

[0024] In some embodiments, the fusion protein has the amino acid sequence of SEQ ID NO:2, or has at least 80% sequence identity thereto. In some embodiments, the fusion protein is encoded by a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1, or has at least 80% sequence identity thereto.

[0025] Some aspects of the disclosure provide an isolated nucleic acid molecule or a construct or vector comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes a fusion protein of the disclosure. In some embodiments, the nucleic acid molecule has an amino acid sequence of SEQ ID NO: 1, or has at least 80% sequence identity to said sequence. In some embodiments, the nucleic acid molecule encodes a polypeptide having an amino acid sequence of SEQ ID NO: 2, or has at least 80% sequence identity to said amino acid sequence.

[0026] In some embodiments, the vector is selected from a viral vector, an mRNA vector, and a DNA vector.

[0027] In some aspects of the present disclosure, there is provided a cell comprising the fusion protein according to any one of claims 1 to 7, or the nucleic acid molecule, construct, or vector according to claim 8 or 9.

[0028] Some aspects of the disclosure provide compositions comprising a fusion protein, a nucleic acid molecule, a construct, a vector, and / or a cell of the disclosure; and a carrier.

[0029] Some aspects of the disclosure provide for the use of the fusion proteins, nucleic acid molecules, constructs, vectors, cells and / or compositions of the disclosure in the preparation of a medicament for preventing and / or treating a viral infectious disease.

[0030] In some aspects of the present disclosure, there is provided a method for preventing and / or treating a viral infection disease, the method comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of a fusion protein, nucleic acid molecule, construct, vector, cell, composition or drug comprising the above of the present disclosure.

[0031] In some aspects of the disclosure, there are provided fusion proteins, nucleic acid molecules, constructs, vectors, cells, compositions and / or drugs of the disclosure, which are used for the prevention and / or treatment of viral infectious diseases.

[0032] In some embodiments, the viral infection is selected from acute viral infections, such as respiratory viral infections and enteric viral infections.

[0033] In some embodiments, the viral infection is caused by one or more viruses selected from the following: coronavirus, influenza virus, rhinovirus, adenovirus, parainfluenza virus, respiratory syncytial virus, coxsackievirus, ecovirus, and novel enteric virus.

[0034] In some embodiments, the fusion proteins, nucleic acid molecules, constructs, vectors, cells, compositions and / or drugs of the present disclosure are administered prophylactically prior to the onset of a viral infection as a prophylactic to prevent the onset of a viral infection or reduce the severity of a subsequent viral infection.

[0035] In some embodiments, the fusion proteins, nucleic acid molecules, constructs, vectors, cells, compositions and / or drugs of the present disclosure are administered as a therapeutic following the onset of a viral infection to reduce the severity of the viral infection and disease.

[0036] In some embodiments, the fusion proteins, nucleic acid molecules, constructs, vectors, cells, compositions and / or drugs of the present disclosure are administered either prophylactically or therapeutically, either continuously or at intervals before and after the onset of viral infection.

[0037] In some embodiments, the formulations of the fusion proteins, nucleic acid molecules, constructs, vectors, cells, compositions and / or drugs disclosed herein are suitable for a mode of administration selected from the following: airway nebulization, nasal drops, sprays, oral administration, intramuscular injection and / or intravenous administration.

[0038] In some embodiments, the fusion proteins, nucleic acid molecules, constructs, vectors, cells, compositions and / or drugs of the present disclosure are suitable for use alone or in combination with other anti-viral, immunological or viral therapies.

[0039] Those skilled in the art can arbitrarily combine the above technical solutions and technical features without departing from the inventive concept and protection scope of the present invention. Based on the contents disclosed herein, other aspects of the present invention will be obvious to those skilled in the art. [Brief description of the drawings]

[0040] The present invention is further described below with reference to the accompanying drawings, the representations of which are only intended to illustrate embodiments of the present invention and are not intended to limit the scope of the present invention. [Figure 1] Construction and in vitro expression of fusion protein expression vector. Figure 1A: Construction map of eukaryotic expression vector pSV1.0 IFNα2-TFF2-Fc, pSV1.0 TFF2-IFNα2-Fc and pSV1.0 2xTFF2-IFNα2-Fc of TFF2 and IFNα2 fusion protein; Figure 1B: Expression of TFF2 and IFNα2 fusion protein with different signal peptides in 293F suspension cell line and CHO-K1 cells; Blank is control cells, Cell is cells, Sup is supernatant, and signal peptides are TFF2, IL-2 signal peptide and tPA2 signal peptide, respectively. [Diagram 2] Validation of TFF2 and IFNα2 fusion protein and its effect on viral protein expression and replication. Figure 2A: Validation of TFF2 and IFNα2 fusion protein after purification by polyacrylamide gel electrophoresis (PAGE); Figure 2B & 2C: Effect of TFF2 and IFNα2 fusion protein on interferon-mediated transmembrane protein 3 (IFITM3) protein expression in lung epithelial cell line A549; Figure 2D & 2E: Effect of TFF2 and IFNα2 fusion protein on influenza virus PR8 replication in lung epithelial cell line A549 in vitro. [Diagram 3]Effect of TFF2 and IFNα2 fusion protein on virus-mediated expression of inflammation-related factors. Figure 3A: Effect of TFF2 and IFNα2 fusion protein on PR8-mediated COX-2 expression; Figure 3B: Effect of TFF2 and IFNα2 fusion protein on PR8-induced IL-6 expression; Figure 3C: Effect of TFF2 and IFNα2 fusion protein on LPS-mediated iNOS expression in ex vivo lung epithelial cell line A549. *** indicates p<0.001. [Figure 4] Protective effects of TFF2-IFNα2-Fc, IFNα2-TFF2-Fc on animals challenged with influenza virus. Figure 4A: TFF2-IFNα2-fc, IFNα2-TFF2-Fc influenza virus challenge and administration model; Figure 4B: Survival rate curve and body weight change curve of mice after PR8 infection (TFF2-IFNα2-Fc); Figure 4C: Survival rate curve and body weight change curve of mice after PR8 infection (IFNα2-TFF2-Fc); Figure 4D: TFF2-IFNα2-Fc, 2xTFF2-IFNα2-Fc influenza virus challenge and administration model; Figure 4E: Survival rate curve of mice after PR8 infection (TFF2-IFNα2-Fc, 2xTFF2-IFNα2-Fc); Figure 4F: Body weight change curve of mice after PR8 infection (TFF2-IFNα2-Fc, 2xTFF2-IFNα2-Fc). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] The present inventors have conducted in-depth research over a long period of time to construct various forms of fusion proteins of TFF2 and IFNα2, and have overcome various technical difficulties by testing, comparing and screening the functions of antiviral, inflammation reduction, animal protection effects, etc., between TFF2 or IFNα2 polypeptide alone and various fusion proteins, to obtain a specific fusion protein form with correct structure and excellent effects. Thus, the present disclosure provides a fusion protein of TFF2 and IFNα2 with a specific structure and composition ratio, and has verified its excellent preventive and therapeutic effects against viral infection diseases. The fusion protein of the present application has excellent antiviral effects and effects of suppressing excessive inflammation, and these effects are significantly superior to those of TFF2 or IFNα2 alone, have synergistic effects, and are significantly superior to those of TFF2 and IFNα2 fusion proteins with other structures and composition ratios.

[0042] Specifically, in the present disclosure, TFF2 polypeptide and IFNα2 polypeptide are fused and expressed in various forms, and the successful expression of the fusion protein is verified by in vitro experiments, and the effect of the fusion protein of TFF2 and IFNα2 in suppressing influenza virus replication and reducing inflammatory factor secretion is measured. In a mouse influenza infection model, it was found that inhalation of the fusion protein of TFF2 and IFNα2 by atomization increases the survival rate of influenza-infected mice and reduces the weight loss of the mice. Among the multiple fusion proteins constructed, the fusion protein in which the TFF2 polypeptide and IFNα2 polypeptide contained are fused at a molecular ratio of 1:1 and the TFF2 polypeptide is located at the N-terminus of IFNα2 has the most excellent effect. Test results show that the antiviral and excessive inflammation suppressing effects of this particular type of fusion protein in vivo and in vivo are significantly superior to TFF2 or IFNα2 alone, have a synergistic effect, and are significantly superior to the fusion proteins of TFF2 and IFNα2 with other structures and formulation ratios.

[0043] More specifically, in order to develop new antiviral drugs and verify their effectiveness in the prevention and treatment of antiviral infections, the inventors constructed eukaryotic expression vectors pSV1.0 IFNα2-TFF2-Fc, pSV1.0 TFF2-IFNα2-Fc and pSV1.0 2xTFF2-IFNα2-Fc, expressed them in 293T cells, verified the expression of the fusion protein by Western Blot, and expressed them in 293F cells, collected the supernatant, and then purified with AKTA pure, identified the purity of the purified protein by Coomassie Brilliant Blue, and measured the protein concentration by BCA. In vitro cell experiments, Western blot was used to detect the effect of the fusion protein of TFF2 and IFNα2 on the replication of influenza virus PR8 in vitro lung epithelial cell line A549, proving that the fusion protein can reduce the replication of the virus.

[0044] Mice were infected with influenza virus H1N1 strain PR8 virus strain, and the weight change and survival rate of mice after PR8 infection were observed. It was found that the TFF2 + IFNα2 fusion protein treatment group could significantly increase the survival rate of mice, reduce weight loss, and improve the symptoms of highly pathogenic influenza infection. Based on this, a preventive experiment of TFF2 + IFNα2 fusion protein was carried out to further verify the preventive and protective effect against viral attack by influenza virus H1N1 strain PR8 strain. These results fully demonstrated that TFF2 + IFNα2 fusion protein exerts important protective effects in acute viral infection damage models induced by respiratory viruses and other factors.

[0045] Since the mechanism of tissue damage caused by other acute viral infections is similar to that of influenza virus infection, the protective effect of the fusion protein of TFF2 and IFNα2 is not limited to respiratory tissue damage caused by influenza virus, but also includes respiratory damage caused by other acute viral infections and enteric diseases that cause acute symptoms such as enteric viruses. In addition, prophylactic administration to disease control personnel and high-risk individuals involved in the epidemic of acute viral infections can effectively reduce the risk and damage to these individuals.

[0046] In other words, based on previous research, the present application further improves and optimizes the method, fusing TFF2 and IFNα2 in a specific form, which can simultaneously exert multi-molecular and multi-functional effects, and is more effective than administering a single drug. Forming a single molecule through fusion can exert a multifunctional effect, while at the same time reducing the complexity of the combination of multiple proteins, improving the therapeutic effect, and exerting a dual effect of antiviral and reducing inflammation. The single molecule TFF2 and IFNα2 fusion protein is used to analyze the antiviral, anti-inflammatory, and protective functions of the fusion protein in viral infection diseases, especially acute viral infection diseases. The specific fusion protein disclosed herein targets the common pathogenic mechanism of acute viral infection, exerts IFNα2 broad-spectrum viral replication suppression function, while suppressing inflammatory cytokine storm by TFF2, promotes the repair of mucosal damage, improves prognosis, and exerts antiviral and repair promotion effects. At the same time, the complementarity between the two fusion components unexpectedly exerts a significantly better effect than the single administration of both. By adjusting the expression ratio of TFF2 and IFNα2 in the fusion protein, it contributes to eliminating the side effects of IFNα2 inducing inflammatory cytokine storm, and helps to achieve the purpose of safe and effective interference.

[0047] In addition, TFF2 is a host small molecule polypeptide, which is highly conserved in different species. For example, the mature human TFF2 molecule is composed of 106 amino acids, has a molecular weight of about 12 kD, contains two symmetric special conserved sequences consisting of about 40 amino acid residues, and contains three intrachain disulfide bonds (cys1-cys5, cys2-cys4 and cys3-cys6) consisting of six cysteine ​​residues, thereby producing a specific and stable cloverleaf structure, which is acid-resistant, heat-resistant, resistant to protease hydrolysis, and has a great advantage in transport. IFNα2 is also a polypeptide secreted by the host and is widely used clinically. Therefore, when TFF2 and IFNα2 are fused and expressed, it not only has clear activity and good safety, but also has high druggability and has high applicability in the prevention and treatment of acute respiratory virus infection and enteric virus infection.

[0048] This disclosure presents the first interference strategy of TFF2 and IFNα2 fusion protein, adopting a three-in-one strategy of antiviral + inflammation suppression + repair promotion, which can be used to prevent and treat acute infectious diseases caused by a wide range of viral infections. The fusion protein can be administered not only systemically but also locally by atomized inhalation, and has a strong targeting, rapid effect, fewer side effects, and higher efficacy. In addition, the product disclosed in this disclosure is low-cost and easy to popularize and apply in economically weak countries and regions, and has high economic value, social value, and political significance as a technical reserve for the country to prevent and manage newly emerging epidemic viral infectious diseases.

[0049] The present disclosure provides a fusion protein of trefoil factor 2 and interferon α2, which has advantages such as inhibiting viral replication, reducing tissue inflammation, reducing tissue damage, and promoting functional repair of lung tissue, and can be used to prepare a drug for the treatment and / or prevention of acute viral infection diseases, and the drug has an obvious improving effect on the prognosis of acute viral infection.

[0050] Compared with the prior art, the present disclosure provides a fusion protein of TFF2 and IFNα2. The inventors experimentally prove that the fusion protein molecule of TFF2 and IFNα2 plays an important role in responding to the epidemic of newly emerging viral infections in the respiratory tract by exerting a protective effect in influenza virus PR8 infection, reducing the morbidity and mortality rate, and alleviating the inflammatory symptoms of respiratory infection, and is particularly beneficial in preventing and treating viral infections and severe infections for which there are no effective therapeutic drugs. The fusion protein of TFF2 and IFNα2 targets the common pathogenic mechanism of acute viral infections, inhibits the inflammatory response, promotes the repair of mucosal tissues, reduces tissue damage, and inhibits the replication function of viruses to exert an antiviral effect. Therefore, the protective effect of the fusion protein of TFF2 and IFNα2 is not limited to respiratory tissue damage caused by influenza virus, but also includes damage caused by other viral infections, and its prophylactic administration to disease control personnel involved in the epidemic treatment of acute viral infections and high-risk individuals can effectively reduce the risk and damage of these individuals.

[0051] All numerical ranges provided herein are intended to explicitly include all numerical values ​​between the endpoints of the range and the numerical range therebetween. Features mentioned in the present invention or in the examples can be combined. All features disclosed herein can be used in any combination, and each feature disclosed in the specification can be replaced with any alternative function that can provide the same, equal, or similar purpose. Thus, unless otherwise specified, the features disclosed are merely generic examples of equivalent or similar functions.

[0052] As used herein, "containing", "having" or "comprising" includes "comprising", "consisting primarily of", "consisting essentially of", and "consisting of"; "consisting primarily of", "substantially of", and "consisting of" are subordinate concepts to "containing", "having" or "comprising".

[0053] As used herein, "mammal" can include humans, primates, rodents (eg, mice, rats, guinea pigs, hamsters), domestic animals or farmed mammals.

[0054] TFF2 and IFNα2 elements As used herein, the term "element" refers to an amino acid sequence that constitutes a part of a fusion protein. The term "unit" refers to a basic part of the element's constituent function. For example, the TFF2 element of a fusion protein may contain one or more consecutive or spaced TFF2 units, each of which produces a desired TFF2-related functional activity.

[0055] As used herein, the terms "TFF2 element" and "TFF2 protein (polypeptide)" are used interchangeably and refer to a native (e.g., mammalian), recombinant or synthetic TFF2 polypeptide sequence that constitutes part of a fusion protein. Native TFF2 is highly conserved among mammals, has a unique and stable cloverleaf structure, and has certain mucosal repair and anti-inflammatory activities. TFF2 polypeptides also include naturally occurring variants and fragments of TFF2 (e.g., splice variants and allelic variants), as well as non-naturally occurring variants that have native TFF2 activity.

[0056] The nucleotide and amino acid sequences of the native TFF2 polypeptide are known, see, for example, GenBank Accession Nos. (human Gene ID: 7032; mouse Gene ID: 21785).

[0057] The TFF2 element herein may comprise the amino acid sequence of SEQ ID NO:8 (human TFF2) or may be encoded by a nucleic acid molecule comprising SEQ ID NO:7, or may comprise the amino acid sequence of SEQ ID NO:12 (mouse TFF2) or may be encoded by a nucleic acid molecule comprising SEQ ID NO:11, or may be a homologous sequence (e.g., homologous sequences can be obtained from databases or alignment software known in the art), mutant or modified form having the same or similar activity as these proteins. For example, the TFF2 polypeptide is selected from: (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:8 or 12 (e.g., the sequence is a polypeptide set forth in SEQ ID NO:8 or 12); or (b) a protein or polypeptide derived from (a) having one or more amino acid substitutions, deletions or insertions in the amino acid sequence defined by (a) and having mucosal repair and anti-inflammatory activity.

[0058] As used herein, the terms "IFNα2 (polypeptide)" and "IFNα2 protein (polypeptide)" are used interchangeably and refer to natural (e.g., mammalian), recombinant or synthetic IFNα2 polypeptides. As discussed in the background section, the structure and function of IFNα2 have been studied and understood in the art. The present application may employ IFNα2 polypeptides known in the art, as well as naturally occurring variants and fragments thereof (e.g., splice variants and allelic variants), and non-natural variants having IFNα2 activity.

[0059] The nucleotide and amino acid sequences of the native IFNα2 polypeptide are known, see, for example, GenBank Accession Nos. (human Gene ID: 3440; mouse Gene ID: 15965).

[0060] The IFNα2 protein herein may be encoded by a nucleic acid molecule that contains the amino acid sequence of SEQ ID NO: 10 (human IFNα2) or contains SEQ ID NO: 9, or the amino acid sequence of SEQ ID NO: 14 (mouse IFNα2) or contains SEQ ID NO: 13, or may be a homologous sequence (e.g., homologous sequences can be obtained from databases or alignment software known in the art) having the same or similar activity as these proteins, mutants or modified forms. For example, the IFNα2 polypeptide is selected from: (a) a polypeptide that contains the amino acid sequence shown in SEQ ID NO: 10 or 14 (e.g., the sequence is a polypeptide shown in SEQ ID NO: 10 or 14); or (b) a protein or polypeptide derived from (a) that has one or more amino acid substitutions, deletions or insertions in the amino acid sequence defined by (a) and has mucosal repair and anti-inflammatory activity.

[0061] The TFF2 polypeptide element and the IFNα2 polypeptide element in the fusion protein of the present disclosure are preferably encoded by a human gene or its homologous or family member. The mutated form of the protein or polypeptide of the present disclosure includes, but is not limited to, one or more (generally 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid deletions, insertions and / or substitutions, and one or more (generally 20 or less, preferably 10 or less, more preferably 5 or less) amino acids added to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar properties generally does not change the function of the protein or polypeptide. It should be noted that the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein or polypeptide, e.g., a fusion protein, which may or may not include an initial methionine residue, that has the desired activity for preventing or treating viral infection.

[0062] Variant forms of a polypeptide include homologous sequences, conservative variants, allelic variants, naturally occurring variants, induced variants, proteins encoded by sequences capable of hybridizing to the protein coding sequence under conditions of high or low stringency. Depending on the host used in a recombinant production protocol, the proteins or polypeptides of the present invention may be glycosylated or non-glycosylated.

[0063] Fusion proteins As used herein, the term "fusion protein" refers to an amino acid molecule that comprises at least one TFF2 polypeptide and at least one IFNα2 polypeptide fused together.The fusion protein of the present disclosure can be produced by recombinant technology from prokaryotic or eukaryotic host (e.g., bacteria, yeast, higher animal, insect, mammalian cell; preferably, eukaryotic host), or can be produced by artificial synthesis, for example, by synthesizing the entire sequence, or by synthesizing fragments and then joining them together.

[0064] The TFF2 peptide in the fusion protein of the present specification can be connected to the N-terminus or C-terminus of the IFNα2 peptide, and preferably, the TFF2 peptide can be connected to the N-terminus, i.e., upstream, of the IFNα2 peptide. The fusion protein of the present specification may contain one or more TFF2 peptides and / or IFNα2 peptides, and for example, the TFF2 peptide and the IFNα2 peptide are fused at a molecular ratio of 5:1 to 1:5, for example, at a molecular ratio of 1:1 or 2:1, and preferably at a molecular ratio of 1:1.

[0065] In a preferred embodiment of the present application, the TFF2 peptide in the fusion protein herein is connected to the N-terminus of the IFNα2 peptide, and the TFF2 peptide and the IFNα2 peptide are fused at a molecular ratio of 1:1.

[0066] The fusion protein of the present application further comprises an Fc region. As used herein, the term "Fc region" or "Fc fragment" refers to an immunoglobulin Fc segment used in the fusion protein. In some embodiments, the Fc region has essentially the same amino acid sequence as a native or mutated immunoglobulin Fc fragment and has essentially the same biological activity as a native Fc fragment. In addition to the CH2 and CH3 regions of an immunoglobulin, the Fc region may further comprise a hinge region. The Fc region may be derived from, for example, IgG or IgA.

[0067] Similar to the function of the Fc segment in a monoclonal antibody, the Fc segment of the fusion protein can extend the half-life of the functional protein in plasma, improve the stability of the molecule, specifically bind to Fc receptors in the body, and exert corresponding biological functions. In addition, the Fc segment can specifically bind to protein A, which simplifies the purification step of the Fc fusion protein, and is of great significance to the development and manufacture of related biological products. The Fc region of the fusion protein herein may contain no mutations or one or more mutations, such as mutations that reduce the activity of antibody-mediated ADCC and CDC, such as amino acid mutations D265A and N297G / N297Q according to the EU numbering system.

[0068] The fusion proteins herein may include a signal peptide, such as an amino acid sequence that functions to direct secretion, localization, and / or trafficking of the fusion protein, typically between 5-50 amino acids in length. In some embodiments, the signal peptide is selected from, for example: tPA2 signal peptide, TFF2 signal peptide, IL-2 signal peptide, bPRL signal peptide, CD33 protein signal peptide, etc.

[0069] The fusion protein of the present invention may contain a marker, for example a marker used for purification, detection, or localization, selected from, for example, a fluorescent marker, a non-radioactive nuclide marker, a biotin-based marker, a phosphorylation modification mark, or a peptide tag.

[0070] Each polypeptide element or peptide unit in an element in a fusion protein of the present specification may be linked by a linker. In the present application, a flexible linker is preferably employed so that there is interaction between the polypeptide elements or peptide units. The linker used in the fusion protein of the present application may contain 2 to 300 amino acid residues, for example, 5 to 100, 10 to 50, or 15 to 3 amino acid residues. An exemplary linker is a glycine linker, for example (G) n or a glycine / serine linker, such as (GS) n , (GGS) n , (GGGS) n , (GGGGS) n or (GGGGGS) n where n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0071] Based on the sequences provided herein and the skills of those skilled in the art, those skilled in the art can easily prepare the fusion proteins of the present invention by various known methods. These methods include, but are not limited to, recombinant DNA techniques, artificial synthesis, etc. [see Murray KM, Dahl SL Ann; Pharmacother 1997 Nov; 31(11)1335-8]. For example, the fusion proteins of the present invention may be produced by direct peptide synthesis using solid-phase techniques, or each fragment of the fusion protein may be chemically synthesized separately and linked by chemical methods to produce a full-length molecule.

[0072] Vectors and hosts The present specification further relates to vectors for producing the fusion proteins and host cells engineered with the vectors.

[0073] The coding sequences of the present invention are used to express or produce recombinant fusion proteins using standard recombinant DNA techniques (Science, 1984;224:1431). Generally, the steps include: (1) transforming or transfecting a suitable host cell with a polynucleotide (or variant) encoding a fusion protein described herein, or with an expression vector containing the polynucleotide; (2) culturing the host cells in an appropriate medium; (3) Isolating and purifying the fusion protein of interest from the medium or cells.

[0074] In the present invention, the terms "vector" and "recombinant expression vector" are used interchangeably and refer to bacterial plasmids, bacteriophages, yeast plasmids, animal cell viruses, mammalian cell viruses or other vectors well known in the art that are capable of replicating and expressing a protein of interest in a host cell.

[0075] Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of the fusion protein and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector further includes a ribosome binding site for translation initiation and a transcription terminator. For example, pSV1.0 vector, pcDNA3.1 vector, pIRES2-EGFP vector, and AdMaxTM expression system can be used herein.

[0076] The expression vectors also preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E. coli.

[0077] A vector containing the appropriate DNA sequence as described above together with an appropriate promoter or control sequence is used to transform an appropriate host cell so as to express the protein or polypeptide. The host cell may be a prokaryotic cell such as a bacterial cell, or a lower eukaryotic cell such as a yeast cell, or a higher eukaryotic cell such as an animal cell. Representative examples are animal cells such as 293F cells, CHO cells, E. coli, Streptomyces, Agrobacterium, and fungal cells such as yeast.

[0078] When the polynucleotide of the present invention is expressed in higher eukaryotic cells, the transcription is enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, that act on promoters to enhance gene transcription. Those skilled in the art will understand how to select appropriate vectors, promoters, enhancers, and host cells.

[0079] By the above method, the fusion protein can be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein may be separated or purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional refolding treatment, treatment with a protein precipitant (salting out method), centrifugation, the AKTA pure method, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other liquid chromatography techniques, and combinations of these methods.

[0080] Drug or kit Provided herein is an article of manufacture containing an effective amount of a fusion protein of the present invention, a vector comprising a coding molecule for the fusion protein, a host cell, and a pharma- ceutical or physiologically acceptable carrier. As used herein, the term "active agent" refers to a fusion protein of the present invention, its coding nucleic acid molecule, a construct or vector comprising said nucleic acid molecule, a host cell, or any of the above compositions.

[0081] In a preferred embodiment, the product herein is used for the prevention or treatment of a disease associated with a viral infection and / or its symptoms. As used herein, the term "comprise" or "having" includes "comprise", "consist essentially of" and "consist of". As used herein, a "pharmaceutical acceptable" ingredient is one that can be applied to humans and / or animals without undue adverse side effects (e.g., toxicity, irritation and allergic reactions), i.e., with a reasonable benefit / risk ratio. As used herein, the term "effective amount" is an amount that is functional or active in humans and / or animals and is tolerated by humans and / or animals.

[0082] As used herein, the term "pharmaceutical acceptable carrier" refers to a carrier used in administering a therapeutic agent, and includes various excipients and diluents. The term refers to some pharmaceutical carriers that are not themselves essential active ingredients, but are not excessively toxic after administration. Suitable carriers are well known to those skilled in the art. A thorough discussion of pharmaceutical acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0083] In the composition, the pharma- ceutically acceptable carrier may contain liquids, such as water, saline, glycerol and ethanol. In addition, these carriers may also contain auxiliary substances, such as fillers, disintegrants, lubricants, glidants, foaming agents, wetting or emulsifying agents, flavorings, pH buffering substances, etc. In general, these substances are non-toxic, inert, and can be formulated in a pharma- ceutically acceptable aqueous medium, usually at a pH of about 5-8, preferably about 6-8.

[0084] As used herein, the term "unit dosage form" refers to the preparation of a composition of the invention into a dosage form required for a single administration for ease of administration, including, but not limited to, a variety of solid formulations (e.g., tablets, lyophilized powders), liquid formulations (e.g., solutions), aerosols, capsules, and sustained release formulations.

[0085] In another preferred embodiment of the present invention, the composition is in a unit dosage form or a multi-dosage form, and the content of the active substance therein is 0.01-2000 mg / dosage, preferably 0.1-1500 mg / dosage, more preferably 1-1000 mg / dosage. In another preferred embodiment of the present invention, 1-6 doses, preferably 1-3 doses of the composition of the present invention are administered daily; most preferably, the dose administered daily is 1 dose.

[0086] The pharmaceutical composition of the present invention may be prepared into various formulations as necessary, and may be administered by such administration methods as airway atomization inhalation, nasal drops, sprays, oral administration, intramuscular injection and / or intravenous administration at a dosage beneficial to the patient determined by a physician according to factors such as the type, age, weight, and general disease state of the patient, and administration method.

[0087] In order to enhance the administration effect, the active substances or products of the present invention may be used in combination with each other, or in combination with other drugs or treatment methods for the prevention and treatment of infectious diseases, especially acute viral infections.For example, when the fusion protein of the present invention is used for the prevention and / or treatment of acute viral infections, other drugs or methods clinically used for the treatment of acute viral infections may be simultaneously or sequentially adopted, including but not limited to the prevention of further injury, regulation of local area function, anti-inflammation, administration of glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs), etc. EXAMPLES

[0088] The present invention will be further described below with reference to specific examples. It should be understood that these examples do not limit the scope of the present invention, but are merely illustrative of the present invention. Those skilled in the art can make appropriate modifications and variations to the present invention, and all of these modifications and variations are within the scope of the present invention.

[0089] For the experimental methods not specified in the following examples, refer to, for example, "Laboratory Guide to Molecular Cloning" (3rd ed., New York: Cold Spring Harbor Laboratory Press, 1989), or use conventional methods in the field, such as following the conditions suggested by the supplier. DNA sequencing is a common method in the art, and tests can also be provided by commercial companies.

[0090] Unless otherwise specified, percentages and parts refer to weight. Unless otherwise defined, all technical and scientific terms used herein are consistent with the meanings known to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described can be applied to the method of the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0091] Example 1: Plasmid construction and design and eukaryotic expression of fusion proteins In this embodiment, first, the gene sequence was cloned based on the amino acid sequence of human TFF2 (e.g., as shown in SEQ ID NO:8) and the amino acid sequence of human IFNα2 protein (e.g., as shown in SEQ ID NO:10), and the recombinant plasmids of different forms were transfected into 293T cells, and then the eukaryotic expression of TFF2 and IFNα2 fusion protein was detected by WB. Then, it was expressed in large quantities in 293F cells, and the expression supernatant was harvested and purified by HiTrap MabSelect SuRe column. The target protein was collected and the purity was identified, and then it was replaced with PBS by ultrafiltration to obtain a highly purified TFF2 and IFNα2 fusion protein. The specific steps were as follows:

[0092] The human TFF2 sequence and interferon α2 were linked with three G4S residues, and TFF2 had different replication clones with a human Fc fragment added to the end (Figure 1A). The correctly sequenced recombinant plasmid was transfected into 293T cells, and the transfection reagent was TurboFect, and the medium was DMEM complete medium (10% FBS and 1% P / S). After 24 hours of culture in a 37°C incubator, the cells were removed, collected, and placed in SDS loading buffer. The cells were collected and placed in an EP tube, washed with PBS buffer, added loading buffer, and heated in a boiling water bath for 10 minutes to denature the protein. After immediate centrifugation, the proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the concentration of the separation gel was 10%. The electrophoresis voltage was 70V, and the time was 30-40 minutes (marked by the start of marker separation). After waiting until bromophenol blue migrated to the separation gel position, the voltage was adjusted to 110V until bromophenol blue migrated to the gum bottom position, and then it was quickly transferred to the membrane and performed at a constant current of 400 mA for 50 minutes. After transferring to the membrane, the front of the PVDF membrane (the side that contacts the gel) was marked, placed in 5% nonfat dry milk, and blocked at room temperature for 2 hours. Next, the primary antibodies (TFF2, Proteintech: 13681-1-AP, 1:1000; IFNα2, SantaCruz, sc-73305, 1:1000; β-actin, ABclonal, AC028, 1:5000) with appropriate dilution ratios were added, diluted with 5% nonfat dry milk, and incubated overnight at 4 °C on a shaker. After washing the membrane with 0.05% PBST, secondary antibodies (goat anti-rabbit (1:5000); goat anti-mouse (1:5000)) were added and diluted with 5% nonfat dry milk in PBST and incubated on a shaker at room temperature for 1 hour. The membrane was then washed, and the membrane was subjected to ECL color development. The PVDF membrane was exposed to light in a quantitative analyzer for 2 minutes, and the color development results were recorded and analyzed.

[0093] As a result (Figure 1B, upper left), the cells expressed large amounts of the fusion protein of TFF2 and IFNα2, and when the TFF2 signal peptide was used, it was found that the fusion protein was mainly expressed in the cells, and the other part was secreted into the supernatant.

[0094] To obtain large amounts of secreted fusion protein, we replaced the signal peptides of TFF2 and IFNα2 recombinant plasmids by replacing their own TFF2 signal peptide with IL-2 signal peptide (Figure 1B, top right) and tPA2 signal peptide (Figure 1B, bottom left, bottom right), and comparing the supernatant secretion of different signal peptides, we found that tPA2 signal peptide promoted the secretion of fusion protein more effectively, so we used tPA2 signal peptide for the expression of fusion protein thereafter.

[0095] Example 2: Purification of the fusion protein and its in vitro antiviral and anti-inflammatory functions To obtain large amounts of recombinant proteins, the correctly sequenced TFF2 and IFNα2 recombinant plasmids were transfected into 293F suspension cells. Specifically, 293F cells were cultured in suspension in SMM 293-TI serum-free medium, supplemented with 1% penicillin / streptomycin antibiotics; 5x10 5 Inoculate fresh medium at a density of 3x10 cells / mL. 6 Experiments were performed or subcultured when the cells reached a density of 1x10 cells / mL or more. The cells were shaken in a cell shaker with 5% carbon dioxide and a rotation speed of 125 rpm / min. The cell density for transient transfection of the fusion protein plasmid was 1x10 cells / mL or more. 6The concentration was 1:1000 cells / mL. Transfection was performed using PEI, and the ratio of DNA:PEI during transfection was 1:3.5. An appropriate amount of plasmid was taken into a 1.5mL EP tube, diluted to 40ng / μL with 150mM sodium chloride, mixed, and then PEI was added. After mixing thoroughly by vortex shaking, the cells were incubated at room temperature for 15-30 min, and the DNA / PEI mix was added to the cells; after 24 hours, a cell clumping prevention agent (Anti-Clumping Agent, Thermo Fisher, 0010057AE) was added at 1:1000; the cells were cultured on a cell shaker for 5-7 days, and the supernatant and cells were collected. WB examination showed that the fusion protein was mainly secreted into the supernatant, so the supernatant was directly purified.

[0096] The fusion protein was purified using the AKTA Pure protein purifier. A HiTrap MabSelect SuRe 1 mL column was used as the column, and all buffers were filtered through a 0.22 μm filter by running the UNICORN software. First, the pump and pipeline were washed with ultrapure water, then connected to the column and washed, and ethanol was washed out with at least 5 volumes of distilled water. The column was balanced by the A1 pipeline using binding buffer (0.02 M sodium phosphate, 0.15 M NaCl, pH 7.2), and filled with elution buffer (0.1 M sodium citrate, pH 3.0) by the B1 pump. Once balanced, the column was loaded through pipeline A1, and the sample was also centrifuged at 12000xg before loading and filtered through a 0.22 μm filter. The column was loaded at a speed of 0.5 mL / min, and the passing liquid was collected at the same time. After loading, the column was washed with binding buffer until the baseline was flat, then switched to B1 and eluted with 100% elution solution. The eluate was collected until the baseline was almost flat, and 100-200 μL of neutralization solution (Tris-HCl, pH 9.0) was added for each 1.5 mL EP tube. The column was eluted and regenerated with 5 CV column volume, washed with 3 CV binding buffer and 5 CV 0.1-0.5 M NaOH, and balanced again with 5-10 CV binding buffer. The column was washed with 20% ethanol and stored at 4 °C. The purified fusion protein was replaced with PBS buffer in a 10 kDa ultrafiltration centrifuge tube. To quantify the purity of the fusion protein of TFF2 and IFNα2, SDS-PAGE was performed according to the above method, and then stained with Quick Blue gel rapid staining solution for 30-60 min and diluted with ddH 2 The cells were washed overnight with 0 (Figure 2A). To quantify the concentration of the fusion protein of TFF2 and IFNα2, a standard curve of BSA was prepared using the BCA quantification kit method, and the concentration of the fusion protein of TFF2 and IFNα2 was quantified based on the optical density (OD) value of the standard. After subpackaging, the cells were frozen and stored at -80°C.

[0097] The interferon activity of the fusion protein was tested in A549 cells. The day before, 1x10 5 Plated at 100 cells / well, added fusion protein, and incubated for 24 h. Wash cells once with PBS, digest with trypsin for 2-3 min, terminate with medium, blow down, wash again with PBS, add 100 μL 1x loading buffer, incubate in boiling water bath for 10 min, and detect interferon-induced transmembrane protein 3 (IFITM3) protein expression by WB (Figure 2B). IFITM3 is an interferon-inducible gene ISG, and its expression is induced by interferon and viruses; the fusion protein stimulates the expression of IFITM3, indicating that the fusion protein has the effect of inducing interferon activity. Fusion of TFF2 and IFNα2 in different orders has different in vitro activity, and IFNα2-TFF2-Fc can induce strong expression of IFITM3, but in the case of TFF2-IFNα2-Fc and 2xTFF2-IFNα2-Fc, the IFITM3 expression level is somewhat weak. Furthermore, in conjunction with subsequent in vitro and in vivo tests, the interferon-inducing activity of the fusion protein was confirmed to be adequate.

[0098] Antiviral experiments of the fusion proteins were performed on A549 cells the day before, by injecting 1x10 5 The cells were plated at 100 μL / well, fusion protein was added 4-6 hours before infection, and the cells were infected at MOI=5 the next day. The cells were infected for 2 hours, washed once with PBS, replaced with DMEM medium with 10% FBS, and incubated for 24 hours. The cells were washed once with PBS, digested with trypsin for 2-3 min, terminated with medium, blown down, washed once more with PBS, added 100 μL 1x loading buffer, incubated in a boiling water bath for 10 min, and then detected by WB for the expression of NP protein (Figure 2C). The results showed that the fusion protein reduced the replication activity of the virus, and the antiviral effect was gradually enhanced with increasing concentration of the fusion protein, but TFF2 alone did not affect the replication activity of the virus.

[0099] COX-2 protein expression was detected by WB (Figure 3A). COX-2 activity in normal tissue cells is extremely low, and when cells are stimulated by inflammation, the expression level in inflammatory cells increases 10 to 80 times higher than the normal level. In the experiment, the action of PR8 virus significantly increased the expression level of COX-2 in A549 cells, and TFF2-IFNα2-Fc and IFNα2-TFF2- Fc fusion proteins also reduced the expression of cyclooxygenase COX-2 in the process of inhibiting PR8 replication, and IFNα2 can also reduce the level of cyclooxygenase COX-2. TFF2 alone cannot inhibit viral replication, and therefore cannot reduce the level of virus-induced COX-2.

[0100] The expression of inflammatory factor IL-6 in the supernatant after PR8 infection was detected by ELISA in A549 cells (Figure 3B). 5 Cells were plated in 12 wells at 10 cells / well and the supernatant was collected at 24 hours. Before detection, the cells were coated with ELISA coating solution (ELISA coating solution: 10 mM sodium carbonate (Na 2 CO 3 ), 30 mM sodium bicarbonate (NaHCO 3), solution pH=9.6, sterilized by filtration through a 0.2 μm membrane filter, and stored at 4°C). 100 μL of capture antibody solution diluted 1:250 was added to each well of the ELISA plate and left at 4°C overnight. The inflammatory factor standard protein is a lyophilized powder. According to the instructions on the label, the lyophilized powder was dissolved in sterile distilled water and diluted 1:1 with the standard diluent. The mother solution concentration after sufficient dissolution was 1000 pg / mL. The standard was gently shaken for 5 min before dilution. The next day, the plate was washed three times with 300 μL / well of washing buffer (PBS with 0.05% Twain-20) and blocked with ELISA blocking solution (PBS with 10% FBS) for 1 hour. Sample loading: 100 μL / well of diluted cytokine standard was added, and a standard concentration gradient: 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, 15.6 pg / mL, 7.8 pg / mL was used to complete the fold dilution against the standard in the imported EP tube. Then, 100 μL / well of sample was added. The sample and standard were incubated for 2 hours, and then washed 5 times with washing buffer. Addition of detection antibody: 100 μL / well of the diluted product was added, and 100 μL of detection antibody + SAv-HRP reagent was added to each well. Incubation was performed at room temperature for 1 hour. The liquid in the well was washed off, the washing buffer was allowed to dwell for 1 min, and then the liquid in the well was discarded, and this was repeated 7 times, and finally the well was dried by fixing it on a filter paper. Color development: 100 μL / well of TMB was added, and the well was incubated at room temperature for 30 min away from light. Termination of reaction: 100 μL / well of termination solution was added immediately to terminate the reaction. Reading of plate: The value was read at 450 nm within 10 min after adding termination solution. The corresponding concentration was found on the coordinate from the absorbance value of the sample.

[0101] As a result, compared with the IFNα2 μg / mL treatment group, the TFF2-IFNα2-Fc fusion protein was able to significantly reduce the IL-6 inflammatory level induced by PR8 at doses of 0.2 and 1 μg / mL, and even at a dose of 5 μg / mL, the IL-6 inflammatory level secreted was equivalent to that secreted when treated with IFNα2. On the other hand, compared with IFNα2, IFNα2-TFF2-Fc was able to induce a high IL-6 inflammatory level and was dose-dependent. IFNα2 itself has an antiviral effect, but its effect also manifests the IL-6 inflammatory level, suggesting that TFF2 and IFNα2 in different orders affect the IL-6 inflammatory level induced by IFNα2. RAW264.7 cells were inoculated with 2.5x10 cells / well into a 12-well plate the day before. 5 Cells were plated at 100 cells / well and fusion proteins were added 4–6 h prior to incubation. After 4 h incubation with fusion proteins at concentrations of 0.2, 1, and 5 μg / mL, LPS 1 ng / mL was added and incubated for 24 h. Cells were washed once with PBS, digested with trypsin for 2–3 min, terminated with medium, washed once more with PBS, added 100 μL 1x loading buffer, incubated in a boiling water bath for 10 min, and the expression of iNOS was detected by WB. In the case of LPS stimulation of RAW264.7 cells, TFF2-IFNα2-Fc produced lower iNOS than IFNα2 (Figure 3C).

[0102] The amount of iNOS may be an indicator of the degree of inflammation in vivo, and the results showed that TFF2-IFNα2-Fc significantly reduced the expression of iNOS compared with IFNα2. IFNα2-TFF2-Fc promoted the expression level of inflammation, and gradually induced an increase in the expression level of iNOS with increasing doses. The structure in which IFNα2 is located at the center of TFF2-IFNα2-Fc affected the function of IFNα2, IFNα2 could induce high iNOS, the structure in which IFNα2 is located at the center could reduce the induced iNOS level, and the structure in which IFNα2 is located at the front of IFNα2-TFF2-Fc did not affect the function of IFNα2, so the induced iNOS level also increased with increasing doses. This result indicates that TFF2-IFNα2-Fc fusion protein can induce appropriate iNOS levels and reduce the risk of excessive inflammatory responses.

[0103] Example 3: Protection and prevention of fusion proteins against viral challenge in animals In this example, influenza virus H1N1 virus strain PR8 (p2 laboratory) was used to infect C57 mice by nasal drop method, and administered 6 hours before infection (Figure 4A). Anesthesia before infection was prepared by preparing a 40 mL solution of 0.5 g tribromoethanol + 1 mL 2-Methyl-2-Butanol + 39 mL water, and anesthetizing the mice at a dose of 300 μL / mouse. Virus challenge was performed at a dose of 1000 TCID50 / mouse. The virus challenge time was set as day 0, and nebulization administration was performed on the 6 h, 2, 4, and 6 days after virus challenge, with a single dose of 0.2, 1, and 5 μg / g. The mice were weighed for 14 consecutive days, and the survival status and survival state of the mice were observed. According to the survival analysis (Figure 4B and Figure 4C), the mice infected with PR8 started to die from day 8 after infection and all died by day 13, while the TFF2-IFNα2-Fc fusion protein could protect about 40% of the mice from influenza death. As shown in Figure 4B and Figure 4C, the weight of the mice infected with PR8 continued to decrease, and by day 10, most of the mice had lost more than 20-30% of their body weight, but the fusion protein group gradually increased their body weight from day 11, and the weight of the IFNα2-TFF2-Fc 0.5 μg / g group continued to decrease. Compared with TFF2-IFNα2-Fc, IFNα2-TFF2-Fc had a lower overall protective effect, which may be due to the reduced protective effect caused by inducing a higher inflammatory response.

[0104] For animal prophylactic experiments with the fusion protein, influenza virus H1N1 virus strain PR8 (p2 in the laboratory) was used to infect C57 mice by nasal drop method, administered 12 hours before infection (Figure 4D). For anesthesia before infection, tribromoethanol 0.5g + 2-Methyl-2-Butanol 1mL + 39 mL of water was used to prepare a 40 mL solution, and mice were anesthetized with 300 μL / mouse. Virus challenge was performed at a dose of 500 TCID50 / mouse. The virus challenge time was set as day 0, and nebulized administration was performed 6 h, 2 days, 4 days, and 6 days after virus challenge, with the doses being 1 μg / g, 1, and 5 μg / g. The mice were weighed for 14 consecutive days, and the survival status (weight change) of the mice was observed. According to the survival analysis (Figure 4E), the mice infected with PR8 started to die on the 7th day after infection, and finally the survival rate of the mice in the PBS group was 30%, but the TFF2-IFNα2-Fc fusion protein protected all the mice from influenza-induced death at a dose of 1 μg / g; as shown in Figure 4F, the weight of the mice infected with PR8 continued to decrease, and by the 7th day, most of the mice had lost more than 10-30% of their body weight, but the fusion protein TFF2-IFNα2-Fc caused a maximum weight loss of about 10% at a dose of 5 μg / g, and almost no weight loss at a dose of 1 μg / g. 2xTFF2-IFNα2-Fc had no protective effect at both the 1 μg / g and 5 μg / g doses, suggesting that the structure of 2xTFF2 in 2xTFF2-IFNα2-Fc affects the function of IFNα2 and thus the protective effect.

[0105] These results demonstrate that the fusion protein TFF2-IFNα2-Fc fusion protein has significantly superior effects in preventing and protecting mice from weight loss and death caused by influenza virus compared with TFF2 alone, IFNα2 alone, and other forms of fusion protein.

[0106] All references mentioned in this application are incorporated herein by reference as if each reference were incorporated solely by reference. It should be understood that based on the above disclosure of the present invention, one skilled in the art may make various changes or modifications to the present invention, and equivalents thereof are also included within the scope defined in the claims appended hereto.

[0107] Attachment: Sequence information [Table 1]

Claims

1. It comprises one or more fusion units, each fusion unit comprising: (a) a trefoil factor 2 (TFF2) element, said TFF2 element comprising a TFF2 peptide or an active fragment thereof; (b) an interferon alpha 2 (IFNα2) element, the IFNα2 element comprising an IFNα2 peptide or an active fragment thereof; However, the TFF2 element and the IFNα2 element are fused at a molecular ratio of 1:1, and in each fusion unit, the TFF2 element is located at the N-terminus of the IFNα2 element. A fusion protein characterized by:

2. The TFF2 peptide or active fragment thereof is derived from a human, a primate, a rodent (e.g., a mouse, a rat, a guinea pig, a hamster), a dog, a cat; and / or The TFF2 peptide or active fragment thereof comprises an amino acid sequence selected from the following: SEQ ID NO:8, SEQ ID NO:12, or an active fragment thereof (e.g., an amino acid sequence having at least 80% sequence identity to SEQ ID NO:8 or SEQ ID NO:12 and having TFF2 activity); and / or The TFF2 peptide or active fragment thereof is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the following: SEQ ID NO:7, SEQ ID NO:11, or an active fragment thereof (e.g., a nucleic acid molecule having at least 80% sequence identity to SEQ ID NO:7 or SEQ ID NO:11 and capable of encoding an active TFF2 peptide). The fusion protein according to claim 1.

3. The IFNα2 peptide or active fragment thereof is derived from a human, a primate, a rodent (e.g., a mouse, a rat, a guinea pig, a hamster), a dog, a cat; and / or The IFNα2 peptide or active fragment thereof comprises an amino acid sequence selected from the following: SEQ ID NO: 10, SEQ ID NO: 14, or an active fragment thereof (e.g., an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 14 and having IFNα2 activity); and / or The IFNα2 peptide or active fragment thereof is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the following: SEQ ID NO:9, SEQ ID NO:13, or an active fragment thereof (e.g., a nucleic acid molecule having at least 80% sequence identity to SEQ ID NO:9 or SEQ ID NO:13 and capable of encoding an active IFNα2 peptide). The fusion protein according to claim 1.

4. The fusion protein further comprises a linker connecting the TFF2 element and the IFNα2 element and / or forming a peptide segment in the elements; For example, the linker is a flexible linker comprising n amino acid residues, where n is an integer between 2 and 300; For example, the linker can be a glycine linker or a glycine / serine linker, e.g., Gn, (GS) ｎ , (GGS) ｎ , (GGGS) ｎ , (GGGGS) ｎ Or (GGGGGS) ｎ where n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The fusion protein according to claim 1.

5. The fusion protein comprises one or more consecutive or spaced TFF2 peptides and / or one or more consecutive or spaced IFNα2 peptides. The fusion protein according to claim 1.

6. the fusion protein further comprises an Fc region, the Fc region containing no mutations or containing one or more mutations that reduce antibody-mediated ADCC and CDC activity, e.g., amino acid mutations D265A and N297G / N297Q according to the EU numbering system; and / or The fusion protein further comprises a signal peptide, for example selected from the group consisting of a tPA2 signal peptide, a TFF2 signal peptide, an IL-2 signal peptide, a bPRL signal peptide, and a CD33 signal peptide; and / or The fusion protein further comprises a marker, for example, a marker used for purification, detection, or localization, and is selected from, for example, a fluorescent marker, a non-radioactive nuclide marker, a biotin marker, a phosphorylation modification mark, and a peptide tag. The fusion protein according to claim 1.

7. The fusion protein has the amino acid sequence of SEQ ID NO:2 or has at least 80% sequence identity thereto; and / or The fusion protein is encoded by a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1 or a nucleic acid molecule having at least 80% sequence identity thereto. The fusion protein according to claim 1.

8. An isolated nucleic acid molecule, characterized in that the nucleic acid molecule encodes a fusion protein according to any one of claims 1 to 7, or a construct or vector comprising said nucleic acid molecule.

9. said nucleic acid molecule has the amino acid sequence of SEQ ID NO:1 or has at least 80% sequence identity thereto; and / or the nucleic acid molecule encodes an amino acid sequence having SEQ ID NO:2 or a polypeptide having at least 80% sequence identity thereto; and / or The vector is selected from a viral vector, an mRNA vector, and a DNA vector. A nucleic acid molecule, construct or vector according to claim 8.

10. A cell comprising a fusion protein according to any one of claims 1 to 7, or a nucleic acid molecule, construct or vector according to claim 8 or 9.

11. A composition comprising a fusion protein according to any one of claims 1 to 7, a nucleic acid molecule, construct or vector according to claim 8 or 9, or a cell according to claim 10; and a carrier.

12. Use of a fusion protein according to any one of claims 1 to 7, a nucleic acid molecule, a construct or a vector according to claim 8 or 9, a cell according to claim 10 or a composition according to claim 11 in the preparation of a medicament for the prevention and / or treatment of a viral infectious disease.

13. The viral infection disease is selected from acute viral infections, such as respiratory viral infections and enteric viral infections; and / or The use according to claim 12, characterized in that the viral infection disease is caused by one or more viruses selected from coronavirus, influenza virus, rhinovirus, adenovirus, parainfluenza virus, respiratory syncytial virus, coxsackievirus, ecovirus, and novel enteric virus.

14. The drug is administered prophylactically, prior to the onset of a viral infection, as a prophylactic drug to prevent the onset of a viral infection or reduce the severity of a subsequent viral infection; The drug is administered after the onset of a viral infection as a treatment to reduce the severity of the viral infection and disease; and / or The drug may be administered either prophylactically or therapeutically, either continuously or at intervals, before and after the onset of viral infection.

13. The use according to claim 12.

15. The dosage form of the drug is suitable for administration by a method selected from the following: inhalation, nasal drops, spray, oral administration, intramuscular injection and / or intravenous administration; and / or The drug is suitable for use alone or in combination with other antiviral, immunological or viral therapies.

13. The use according to claim 12.

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