CN113975299A - Method for preventing and treating respiratory infectious diseases by using respiratory epithelial cell membrane and application - Google Patents

Abstract

The present disclosure relates to methods and uses for preventing and treating respiratory infectious diseases using respiratory epithelial cell membranes. In particular, the present disclosure provides the use of a respiratory epithelial cell membrane, wherein the cell membrane may be unmodified or modified (e.g., modified to overexpress a receptor on the surface of the cell membrane), in the manufacture of a product for preventing and/or treating infection by a pathogen of a respiratory infectious disease, and corresponding products. The respiratory epithelial cell membrane and related products thereof can be widely used for preventing and treating respiratory infectious disease pathogen infection, and the modified cell membrane can play a more effective role in preventing and treating specific pathogen infection.

Description

Method for preventing and treating respiratory infectious diseases by using respiratory epithelial cell membrane and application

Technical Field

The present invention belongs to the field of biotechnology and medicine. In particular to a method for preventing and treating pathogen infection of respiratory infectious diseases by using respiratory epithelial cell membranes and application thereof.

Background

Respiratory infectious diseases refer to infectious diseases caused by invasion of pathogens (such as viruses, bacteria, fungi, mycoplasma, chlamydia, etc.) from respiratory infections such as nasal cavities, pharynx, larynx, trachea, bronchi, and lungs of the human body.

The new, sudden and severe infectious diseases caused by the infection of respiratory infectious disease pathogens seriously threaten the health and social stability of people and seriously create regional communication and economic construction, wherein the most representative diseases comprise: coronaviruses such as SARS (Severe Acute Respiratory Syndrome), SARS (Middle East Respiratory Syndrome, MERS) in 2012, and novel Coronavirus (Severe Acute Respiratory Syndrome Coronavir 2, SARS-CoV-2) in 2019; and highly pathogenic Influenza viruses (Influenza viruses), such as 2009H1N1 "swine flu", 2013 human infection H7N9, and the like. Other pathogens (including viruses, bacteria, fungi, mycoplasma, chlamydia, etc.) transmitted through the respiratory tract, such as measles virus, respiratory syncytial virus, mumps virus, meningococcus, tubercle bacillus, etc., also present serious public health challenges worldwide.

Currently, specific or universal vaccines against pathogens of major respiratory infectious diseases are still under development to obtain more effective therapeutic vaccines or drugs. Traditional anti-pathogen drugs such as Interferon (Interferon, IFN) or monoclonal neutralizing antibodies have great limitations in clinical treatment, for example, the application of IFN α is likely to aggravate pulmonary inflammatory response, while antibody drugs have a single target and are likely to promote pathogen mutant strains to escape immune response. In addition, the existing antibody has insufficient broad spectrum and is difficult to effectively deal with the epidemic situation of the new emergent infectious disease. Therefore, the development of novel and universal biological products and medicines for preventing and treating respiratory infectious disease pathogens is of great significance.

Respiratory infectious disease pathogen (e.g., virus) specific receptors are widely expressed on the surface of human respiratory tissue cell membranes. For example, the primary condition for virus entry and determinants of viral tissue tropism and pathogenesis are virus-specific receptors on the cell membrane surface of respiratory tissues. The main membrane protein of the virus can specifically recognize virus receptor molecules on the surface of a target cell membrane, so that the virus receptor molecules can adsorb and invade target cells, and the replication cycle of the virus receptor molecules is completed by utilizing a replication system of host cells. Therefore, the method cuts off the 'action connection' between the virus membrane protein and the host cell receptor molecule, and is expected to block the invasion of the virus.

Target cell membrane surface receptor molecules have been identified for a number of common important respiratory infectious agents, such as: receptors for influenza virus, mumps virus include sialic acid or sialic acid modified glycoproteins; the receptor of highly pathogenic coronaviruses (SARS-CoV and SARS-CoV-2) is Angiotensin converting enzyme (ACE 2); the receptor for middle east coronavirus (MERS-CoV) is the CD26 (also known as Dipeptidyl peptidase-4, DPP4) molecule; the receptor for human coronavirus (hCov-229E) is CD13 peptidase (APN); measles virus utilizes CD46 and SLAM family molecules as receptors; respiratory syncytial virus utilizes CX3CR1 as a receptor molecule; chlamydia pneumoniae enters target cells by binding IGF-2 molecules. Specific receptors for pathogens of respiratory infectious diseases may include more than one molecule, frequently occurring co-receptor molecules as well as multi-receptor molecules. Thus, the surface of the epithelial cell membrane of the human respiratory tract may not only have the identified receptor molecules, but may also have other receptor molecules that are unknown and have not been identified.

Meanwhile, a plurality of anti-infective protein molecules (such as anti-virus infective protein molecules, for example, interferon-induced transmembrane protein IFITM1) and molecules for regulating the immune response of body cells (such as TNF-alpha receptor and interferon receptor alpha/beta) are also present on the cell membrane of the epithelial cells of the respiratory tract. These protein molecules play an important role in host resistance to infection by respiratory pathogens.

U.S. patent application publication nos. US20130337066a1, US20160136106a1, US20170274059a1, and US20180169027a1 describe the use of cell membranes (including bacterial envelopes and mammalian cell membranes) to coat nanomaterials (containing specific drugs) in vitro as drug delivery vehicles for vaccine, tumor, infectious disease, and the like. The mammalian cell membrane mainly refers to human erythrocyte membrane, immune cell membrane and platelet membrane. The main principle involved is that the anti-tumor and other drugs contained in the inner core of the nano-particle are delivered to specific tissue cells in a targeted manner through the cell membrane wrapped by the outer layer of the nano-particle, so that the purpose of targeted delivery of the drugs is achieved. However, these documents do not disclose cell membranes and receptors on their surfaces as means for inhibiting viral infection.

In view of the above, there remains a great need in the art to develop methods and products that are effective in preventing and/or treating infection by respiratory infectious agents.

Disclosure of Invention

The present disclosure provides a use, method and product effective for the prevention and/or treatment of respiratory infectious diseases, preferably with a broad spectrum and/or specificity. The uses, methods and products of the present disclosure employ unmodified or modified respiratory epithelial cell membranes that stably express, for example, pathogen-specific receptor molecules, immunomodulatory molecules, and/or anti-infective molecules (e.g., molecules that are resistant to viral infection) to inhibit respiratory pathogens, which can be used to prevent and treat infection and transmission of respiratory pathogens.

In a first aspect of the present disclosure, there is provided the use of a cell membrane of a respiratory epithelial cell in the manufacture of a product for the prevention and/or treatment of infection by a respiratory infectious disease pathogen.

In some embodiments, the pathogen is transmitted through the respiratory tract.

In some embodiments, the pathogen acts on respiratory epithelial cells to effect invasion and/or infection.

In some embodiments, the pathogen is selected from the group consisting of: respiratory infectious diseases virus, bacteria, fungi, mycoplasma, chlamydia, etc.

In some embodiments, the airway epithelial cells are selected from the group consisting of: lung epithelial cells, trachea epithelial cells, bronchial epithelial cells, nasopharyngeal epithelial cells, for example, the epithelial cells selected from A549 cells, Calu-1 cells, Calu-3 cells, BEAS-2B cells or NP69 cells, etc.

In some embodiments, the source of the respiratory epithelial cells is mammalian, e.g., human, rat, mouse, preferably human.

In some embodiments, the cell membrane is an unmodified cell membrane or a modified cell membrane, e.g., the cell membrane is a cell membrane of a wild-type respiratory epithelial cell, a genetically engineered cell membrane, a chemically modified cell membrane.

In some embodiments, the cell membrane is a cell membrane fragment.

In some embodiments, the modified cell membrane is selected from the group consisting of: a cell membrane modified to have overexpressed respiratory infectious disease pathogen cell surface receptors, immunomodulatory molecules, and/or antipathogenic molecules thereon; a cell membrane modified to express thereon a respiratory infectious disease pathogen cell surface receptor, immunomodulatory molecule, and/or antipathogenic molecule not present in wild type; or any combination of the above.

In some embodiments, the cell surface receptor, immunomodulatory molecule, and/or antipathogenic molecule is a full-length sequence, an active fragment thereof, a fusion protein expressed in fusion with other molecules; ways of fusion expression include, but are not limited to, fusion with the Fc sequence of an antibody, fusion with the variable region of an antibody to target an immunogen to a particular cell or region, fusion with a different signal peptide, fusion with a cytokine, and the like.

In some embodiments, the cell surface receptor is one or more selected from the group consisting of: sialic acid or sialic acid modified glycoproteins, angiotensin converting enzyme 2(ACE2), CD26 molecules, CD13 peptidase (APN), CD46, SLAM family members, CX3CR1 molecules, IGF2 molecules, and the like.

In some embodiments, the immunomodulatory or anti-pathogenic molecule is one or more selected from the group consisting of: IFITM1, IFN-k, IL-7, IL-15, IL-21, IL-6 receptor, TNF-a receptor, IL-1 receptor, class I interferon receptor or subunit thereof and toxin receptor.

In some embodiments, the respiratory infectious disease pathogen is one or more selected from the group consisting of: influenza viruses (e.g., H1N1, H3N2, H7N9, H5N1, etc.), coronaviruses (e.g., SARS-CoV-2, MERS-CoV), mumps virus, measles virus, respiratory syncytial virus, Nipah virus, human metapneumovirus, and human Chlamydia pneumoniae, etc.

In some embodiments, the product is a medicament.

In some embodiments, the product is in a form suitable for administration as follows: nasal drops, spray, aerosol inhalation, or any combination thereof.

In one aspect of the present application, there is provided a method of preparing a cell membrane and/or product as described herein, comprising:

(a) providing airway epithelial cells, wherein said cells are modified or unmodified; and

(b) isolating the cell membrane of the cell to obtain an unmodified cell membrane or a modified cell membrane;

(c) optionally, the separated cell membrane is manufactured into a product, such as directly into a drug, coated or coupled to a carrier material (e.g., a biodegradable nanomaterial) to form a drug (e.g., a nanoparticle drug).

In some embodiments, the cells are engineered to overexpress a cell surface receptor, immunomodulatory molecule, and/or antipathogenic molecule of a respiratory infectious disease pathogen or to express a cell surface receptor, immunomodulatory molecule, and/or antipathogenic molecule of a respiratory infectious disease pathogen that is not present in wild-type.

In some embodiments, the cells are engineered with lentiviral vectors, plasmid vectors, poxvirus vectors, adenoviral vectors, adeno-associated viral vectors, herpes simplex viral vectors, CMV vectors, such that the cell surface receptors, immunomodulatory molecules, and/or anti-pathogenic molecules are highly expressed on the surface of the epithelial cell membrane of the respiratory tract.

In another aspect of the present application, there is provided a product comprising a cell membrane of a respiratory epithelial cell as described herein or a cell membrane prepared by a method as described herein.

In other aspects of the present disclosure, there is also provided a method for preventing and/or treating infection by a respiratory infectious disease pathogen or a condition associated therewith, the method comprising administering to a subject in need thereof an effective amount of a respiratory epithelial cell membrane or product as described above.

In other aspects of the disclosure, there is also provided a cell membrane of a respiratory epithelial cell or a product comprising the same, for use in the prevention and/or treatment of infection by a respiratory infectious disease pathogen or a condition associated therewith.

Any combination of the above-described solutions and features may be made by those skilled in the art without departing from the spirit and scope of the present invention. Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

The present invention will now be further described with reference to the accompanying drawings, wherein the showings are for the purpose of illustrating embodiments of the invention only and not for the purpose of limiting the scope of the invention.

FIG. 1: successful construction of ACE2-a549 cell line stably and abundantly expressing ACE2 protein on cell membrane:

a, drawing: western Blot (WB) results showed that ACE2 was abundantly expressed;

b, drawing: the method is characterized in that the ACE2-A549 cells are stained by SARS-CoV-2 receptor binding domain protein RBD with fluorescent labels, and flow cytometry results show that a large amount of functional ACE2 protein is expressed on the surface of the A549 cell membrane.

FIG. 2: a protein immunoblotting (WB) experiment shows that ACE2-A549 cell membrane is successfully prepared by separation. It is shown that Epidermal Growth Factor Receptor (EGFR) and ACE2 proteins, which are cell membrane surface proteins, can be detected in the cell membrane fraction, while β -actin (β -actin), which is a cytoplasmic protein fraction, is hardly detected. Whereas in the cytoplasmic fraction only β -actin is detected, but not the cell membrane proteins EGFR and ACE 2.

FIG. 3: the A549 cell membrane fraction is shown to have inhibitory effect on the infection of the novel coronavirus, pseudovirus. The figure shows that the wild type A549 cell membrane component can effectively inhibit the infection of the new coronavirus, the cell membrane component highly expressing ACE2 has stronger inhibiting effect on the infection of the new coronavirus, and the half Inhibition Concentration (IC) of two cell membranes₅₀) More than two times different.

FIG. 4: shows that the wild type A549 cell membrane component can effectively inhibit the target cells from being infected by influenza A virus H1N1(a picture and b picture) and H7N9(c picture and d picture); the results of Western immunoblotting of a picture and a picture c show that the cell membrane component has a dose-dependent relation with the inhibition effect of influenza A virus H1N1 and H7N9, and the cell membrane component of 500 mug/ml can effectively inhibit virus replication; the b picture and the d picture reflect relative expression values between the expression level of the viral protein and the expression level of the cytoskeletal protein in the a picture and the c picture respectively, and finally are converted into expressions of the inhibition rate of the viral infection.

The labels 1-5 in FIG. 4 correspond to: 1. no infected control; 2. PBS treated control; 3. wt-A549 membrane fraction 500. mu.g/ml; 4. wt-A549 membrane fraction 250 μ g/ml; 5. wt-A549 membrane fraction 125. mu.g/ml.

Detailed Description

The present application provides techniques, methods and products for the broad-spectrum or specific prevention and/or treatment of respiratory infectious diseases. In particular, provided herein are technical solutions for preventing and/or treating respiratory infectious diseases directly using cell membrane fractions of wild-type respiratory epithelial cells. The application further provides a technical scheme for preventing and treating the infection and transmission of pathogens of respiratory infectious diseases by adopting respiratory epithelial cell membranes and components thereof which stably and highly express the specific receptor molecules and/or immunoregulatory molecules of the pathogens (such as viruses).

More specifically, the application discloses a broad-spectrum or specific prevention and treatment technology for respiratory infectious pathogen infection (such as influenza A virus H1N1/H7N9 and coronavirus SARS-CoV-2). In some embodiments, respiratory infectious agent (e.g., virus) receptor proteins (e.g., ACE2) or other anti-pathogen molecules and immunomodulatory molecules are stabilized and expressed in large amounts on the cell membrane of human respiratory epithelial cells (e.g., a549) to produce cell membranes and fractions thereof containing the receptor and other anti-pathogen molecules; cell membrane biologics containing these protein molecules are used to competitively bind to pathogen membrane proteins, thereby blocking infection of target cells by pathogens. Or the immune regulatory molecule directly or indirectly participates in the immune response against pathogens, thereby playing a role in preventing and treating the infection of pathogens of respiratory infectious diseases. The application focuses on the technical method for preventing and treating the pathogen infection of the respiratory infectious disease by using a wild-type respiratory epithelial cell membrane component or a cell membrane component stably expressing a virus receptor protein and an antiviral protein molecule in a large amount and a preparation containing the cell membrane.

The respiratory epithelial cell membrane and products thereof provided herein can serve to prevent and/or treat respiratory infectious diseases and symptoms thereof caused by pathogens by competitively binding to the pathogens, including pathogens that originally invade and/or pathogens that are further produced after replication or reproduction in vivo.

All numerical ranges provided herein are intended to expressly include all numbers between the end points of the ranges and numerical ranges there between. The features mentioned in the present disclosure or the features mentioned in the embodiments can be combined. All the features disclosed in this specification may be combined in any combination, and each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.

As used herein, "about" in the context of a value or range means ± 10% of the recited or claimed value or range.

It is to be understood that when ranges of parameters are provided, the invention likewise provides all integers and decimals thereof within the ranges. For example, "0.1-2.5" includes 0.1, 0.2, 0.3, etc. up to 2.5.

As used herein, "comprising," having, "or" including "includes" comprising, "" consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … …; "consisting essentially of … …", "consisting essentially of … …", and "consisting of … …" are subordinate concepts of "comprising", "having", or "including".

Cell membrane of respiratory epithelial cell and surface molecule thereof

As used herein, respiratory epithelial cells refer to cells distributed or isolated from the epithelium of respiratory tissue, which is typically a major component of infection by pathogens of respiratory infectious diseases. The respiratory tract is the path through which the airflow passes during breathing and comprises: upper respiratory tract, e.g., nose, pharynx, and larynx; and the lower respiratory tract, such as the trachea, bronchi and lungs. The airway epithelial cells include, but are not limited to, epithelial cells of airway tissues as described above, and may be selected from, for example, A549 cells, Calu-1 cells, Calu-3 cells, BEAS-2B cells, NP69 cells, and the like.

The cell membrane of the respiratory epithelial cell (or "cell membrane of the present application" or "cell membrane preparation") refers to a cell membrane or component thereof isolated from the respiratory epithelial cell, which may include cell membrane fragments (i.e., non-intact cell membranes).

As used herein, the cell membrane of the respiratory epithelial cell may be a natural cell membrane (i.e., not artificially modified), also referred to as a wild-type cell membrane, a cell membrane modified to have an overexpressed pathogen cell surface receptor, immunomodulatory molecule, and/or antipathogenic molecule thereon, or a cell membrane modified to express a pathogen cell surface receptor, immunomodulatory molecule, and/or antipathogenic molecule thereon that is not present in the wild-type; or any combination of the above.

The cell membrane carries various molecules that interact directly or indirectly with pathogens that act on the epithelial cell membrane of the respiratory tract, such as viral cell surface receptors, immunomodulatory molecules, and/or antiviral molecules, either known or unknown, alone or conjugated, complexed or fused molecules. For example, the molecule can be a full-length sequence, an active fragment thereof, a fusion protein formed by fusion expression with other molecules.

Pathogen cell surface receptors include, but are not limited to, one or more selected from the group consisting of: sialic acid or sialic acid modified glycoproteins, angiotensin converting enzyme 2(ACE2), CD26 molecules, CD13 peptidase (APN), CD46, SLAM family molecules, CX3CR1, and IGF2 molecules. Anti-antigen peptide molecules or immunomodulatory molecules include, but are not limited to, one or more selected from the group consisting of: IFITM1, IFN-k, IL-7, IL-15, IL-21, IL-6 receptor, TNF-a receptor, IL-1 receptor, class I interferon receptor or subunit thereof and toxin receptor.

Pathogen-specific receptor molecules described herein include, but are not limited to, receptor molecules that have been reported, but also include potential co-receptor molecules present on the epithelial cell membrane of the respiratory tract that are not reported.

Various methods can be used to engineer airway epithelial cells. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. For example, cells are engineered with lentiviral vectors, plasmid vectors, poxvirus vectors, adenoviral vectors, adeno-associated viral vectors, herpes simplex viral vectors, CMV vectors.

For example, an ACE2 receptor molecule may be expressed on the epithelial cell membrane of the respiratory tract, and the ACE2 molecule may have an amino acid sequence as shown in SEQ ID NO. 3, or an amino acid sequence with more than 90% homology to SEQ ID NO. 3, or may be encoded by a nucleic acid molecule comprising the sequence shown in SEQ ID NO. 1 or encoded by a nucleic acid molecule with more than 90% homology to the sequence shown in SEQ ID NO. 1. It was confirmed that the respiratory epithelial cell membrane stably highly expressing ACE2 molecule on the cell membrane has excellent infection-inhibiting effect on respiratory viruses (such as SARS-CoV and SARS-CoV-2) which achieve infection by binding ACE2 receptor molecule.

The technical method described in the present application includes, but is not limited to, biotechnologically expressing a membrane protein gene directly encoding a pathogen-specific receptor molecule in respiratory epithelial cells, encoding a corresponding receptor protein, and biotechnologically allowing other genes or DNA/RNA elements that promote the expression of the corresponding receptor protein gene to function in cells to promote the expression of the corresponding pathogen receptor protein.

The technical method comprises the steps of stably and highly expressing a specific virus receptor molecule on an epithelial cell membrane of a respiratory tract, and simultaneously expressing two or more virus receptors and antiviral molecules on the epithelial cell membrane of the respiratory tract so as to achieve the aim of broad-spectrum prevention and treatment of the respiratory infectious diseases.

The cell membrane of the respiratory epithelial cell can be separated by a conventional method (for example, by using a commercially available cell membrane preparation kit), and the interference of intracellular organelles, nuclei and other components can be eliminated. For example, cells can be lysed and their membrane fraction isolated (e.g., by centrifugation).

Medicament comprising cell membrane

Also provided herein is a product comprising a cell membrane of a respiratory epithelial cell of the present disclosure. In some embodiments, the product can be a pharmaceutical composition (e.g., a pharmaceutical product) comprising a cell membrane of a respiratory epithelial cell of the present disclosure.

The products herein may comprise an effective amount of a cell membrane of a respiratory epithelial cell as described in the present disclosure, and a pharmaceutically acceptable carrier. As used herein, the terms "active agent" or "active agent of the invention" are used interchangeably to refer to the cellular membrane (including components or fragments thereof) of the respiratory epithelial cells of the present disclosure.

In embodiments of the present disclosure, the products herein are useful for the prevention and/or treatment of diseases or conditions associated with infection by pathogens of respiratory infectious diseases. For example, the products of the invention may be used for the prevention and/or treatment of respiratory viral infections.

As used herein, the term "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. As used herein, the term "effective amount" refers to an amount that produces a function or activity in and is acceptable to humans and/or animals.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not essential active ingredients per se and are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences, Mack pub.Co., N.J.1991.

Pharmaceutically acceptable carriers in the compositions may comprise liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances, such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers. Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8.

The cell membrane active substance in the product of the present disclosure may account for 0.001 to 99.9 wt%, such as 1 to 95 wt%, 5 to 90 wt%, 10 to 80 wt% of the total weight of the product. The balance of substances such as pharmaceutically acceptable carriers, other additives and the like.

As used herein, the term "unit dosage form" refers to a form of the product of the present invention that is formulated for single administration as a convenient administration, including, but not limited to, various sprays, liquids, solids (e.g., tablets), and the like.

It is understood that the effective dose of active airway epithelial cell membrane used may vary with the severity of the subject to be administered or treated. The specific condition is determined according to the individual condition of the subject (e.g., the subject's weight, age, physical condition, desired effect), and is within the judgment of a skilled physician.

In addition, the products herein may also contain other active substances for the prevention, amelioration and/or treatment of infectious diseases of respiratory pathogens, which may include, but are not limited to: antibiotics (including beta-lactams (penicillins and cephalosporins), aminoglycosides, tetracyclines, chloramphenicols, macrolides, antifungal antibiotics, antitubercular antibiotics) are commonly used clinically.

Applications of

Also provided herein is a method for preventing and/or treating infection by a respiratory infectious disease pathogen and/or symptoms thereof, comprising: at least one administration of a prophylactically and/or therapeutically effective amount of one or more cell membranes and/or products of the present disclosure.

The application aims at preventing and treating pathogens of respiratory infectious diseases, including but not limited to influenza viruses (influenza A virus and influenza B virus), coronaviruses (SARS-CoV-2, SARS-CoV, MERS-CoV), measles virus, respiratory syncytial virus, mumps virus and the like, and also includes other pathogens (viruses, bacteria, fungi, mycoplasma, chlamydia and the like) transmitted through respiratory tract.

The subject to which the products of the present application are applied may be a human or other mammal, e.g., a non-human primate such as chimpanzees and other apes and monkey species; livestock such as cattle, sheep, pigs, and horses; domestic mammals such as dogs and cats; laboratory animals, including rodents such as mice, rats and guinea pigs, and the like. The terms "mammal" and "animal" are included in this definition and are intended to encompass adult, juvenile, and newborn individuals.

Providing the combination product herein in the form of a pharmaceutical pack or kit may, for example, be packaged with one or more pharmaceutical compositions or medicaments herein or one or more of its ingredients in one or more containers, for example in a sealed container such as an ampoule or sachet indicating the amount of composition. The compositions can be provided as a liquid, sterile lyophilized powder, or anhydrous concentrate, and the like, which can be diluted, reconstituted and/or formulated with an appropriate liquid (e.g., water, saline, etc.) just prior to use to obtain the appropriate concentration and form for administration to a subject.

Administration regimens that may be employed include, but are not limited to: administration modes in respiratory tract, such as atomization, nasal drops, spray and the like; systemic administration, such as intravenous injection, etc. In a preferred embodiment of the present application, the product of the present application is administered via the respiratory tract. In this respect, the product of the application does not need to be additionally added with specific drug delivery materials such as other drugs wrapped by nano materials, and does not need to be injected into the body such as intravenous injection, so that the cost can be obviously reduced, the medication safety is improved, and the method is a broad-spectrum and specific technical method for preventing and treating respiratory infectious diseases.

Examples

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art can make appropriate modifications and alterations to the present invention, which fall within the scope of the invention.

The experimental procedures for the conditions not specified in the examples below can be carried out by methods conventional in the art, for example, by referring to the molecular cloning, A Laboratory Manual, New York, Cold Spring Harbor Laboratory Press, 1989 or according to the conditions recommended by the supplier. Methods for sequencing DNA are conventional in the art and tests are also available from commercial companies.

Unless otherwise indicated, percentages and parts are by weight. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Example 1: construction of lung epithelial cell line ACE2-A549 for stably expressing ACE2 protein

The lung epithelial cell line A549 (purchased from ATCC # CCL-185) is derived from human non-small cell lung cancer epithelial cells and is a major in vitro cell model for studying infection by pathogens of respiratory infectious diseases.

Human ACE2 gene (SEQ ID NO:1) was synthesized in vitro by gene synthesis technology, ACE2 gene was inserted into lentiviral vector Phage-puro (adddge #118692), and 293T cells were co-transfected with packaging plasmid Pspax2 (adddge #12260) and envelope plasmid PMD2.G (adddge #12259), and lentiviral supernatants were harvested after 48 hours. Infecting A549 cells with recombinant ACE2 lentivirus, centrifugally infecting for 2 hours, continuously culturing for 24 hours, screening the A549 cells expressing ACE2 by puromycin, continuously adding medicine for screening for 14 days, and finally obtaining ACE2-A549 cells.

The expression of ACE2 gene in A549 cells was verified by western blotting (FIG. 1a), and the expression of ACE2 on A549 cells was verified by flow cytometry (FIG. 1 b). In Western blotting experiments, the dilution ratio of human ACE2 antibody is 1:1000 (rabbit polyclonal antibody abcam # ab15348), and the dilution ratio of beta-actin (beta-actin) antibody is 1:3000 (rabbit monoclonal antibody abcam # ab 179467). In the flow cytometry experiment, a fusion protein (RBD-mFC, Novoprotein # DRA32) of a SARS-CoV-2 receptor binding domain RBD marked by a fluorescent dye (Alexa Fluor488, Invitrogen # A30006) and mouse FC is incubated with ACE2-A549 for 20 minutes at normal temperature, and then the expression of ACE2 on cell membranes is detected by a flow detector.

The result shows that wild type A549 does not express ACE2 protein, and the ACE2-A549 cell line constructed by the method can stably and efficiently express ACE 2. The above results demonstrate the successful construction of an a549 cell line stably and efficiently expressing ACE 2.

Example 2: separation and preparation of cell membrane

In this example, the following three cell lines were separately subjected to cell membrane separation and preparation: wild type A549(Wt-A549), empty vector control A549(vector-A549), and A549 stably expressing ACE 2(ACE 2-A549).

Cell membrane separation kit (Thermo fisher scientific #89842) was used to extract, separate, and prepare cell membrane samples. The brief experimental procedure is as follows: will be 4X 10⁶The cells were plated on 10 cm cell culture dishes, and a total of 5 dishes were prepared to give a total of 2X 10⁷Number of individual cells. After conventional culture in a 37 ℃ cell incubator for 12 hours, the cells were washed three times with cold PBS buffer and collected by scraping. After washing the cells twice with the cell wash solution in the kit, the cells were treated with the permeabilization buffer for 10 minutes to release the cytoplasmic components. After centrifugation at 16,000g for 15 minutes, the supernatant was removed, the pellet was resuspended in solubilization buffer, the cell membrane fractions were redissolved in solution, and treated at 4 ℃ for 30 minutes. After centrifugation at 16,000g for 15 minutes, the supernatant containing the cell membrane fraction was transferred to a new EP tube. Desalting was performed using an ultrafiltration tube (Merck, # UFC9003-3 KD). The cell membrane sample was diluted with pre-cooled PBS each time and centrifuged at 4,500g for 30 minutes at 4 ℃ until the dilution ratio was 1000-fold for desalting.

The protein concentration in the cell membrane fraction samples was quantified using standard BCA methods (for protein quantification in subsequent experiments) and the cell membrane fraction separation efficiency was verified using western blotting. The successful preparation of cell membrane separation was verified by examining the expression of Epidermal Growth Factor Receptor (EGFR), ACE2, a protein related to cell membrane components, and the expression of β -actin, a cytoplasmic component marker (β -actin) (fig. 2).

Example 3: inhibition of SARS-CoV-2 infection in vitro using cell membranes or components thereof

This example uses the coronavirus pseudovirus system to detect pseudovirus infected cells at the cellular level. 293T cells were co-transfected with a novel coronavirus (SARS-CoV-2) spinous process protein gene (Spike, SEQ ID NO:4, reference GenBank: QHD43416.1 expression plasmid pcDNA3.1-Spike (Spike whole gene synthesis by Shanghai Czert Biotechnology Co., Ltd., cloned into pcDNA3.1 expression vector) and an HIV-1 backbone plasmid with luciferase reporter gene (pNL4-3-Luc- Δ Env, NIH AIDS Reagent Program #3418), and cell culture supernatants containing coronavirus pseudoviruses were harvested 48 hours later.

293T cells expressing ACE2 (the construction method of an ACE2-293T cell line is the same as that of ACE2-A549) are infected by supernatants containing pseudoviruses, and if the target cells are successfully infected by the pseudoviruses, the infection efficiency of the pseudoviruses can be reflected by detecting the expression level of a reporter gene luciferase in the infected cells. The method comprises the following steps: the cell membrane sample prepared by separation in example 2 was serially diluted twice in the cell completion medium, and 50. mu.l of the cell membrane diluted sample was mixed with 50. mu.l of a solution containing 200TCID₅₀The coronavirus pseudoviruses of (1) were mixed, incubated at 37 ℃ for 1 hour, added to a 96-well cell culture plate containing 20,000 ACE2-293T cells, and the expression level of luciferase was measured after 48 hours of culture.

The results show that wild type A549 and ACE2-A549 cell membrane components can effectively inhibit the target cells infected by the novel coronavirus, the inhibition effect of the cell membrane components after over-expression of ACE2 protein is more obvious (figure 3), and the half inhibition concentration IC is₅₀About 2 times lower than the wild type.

Example 4: in vitro inhibition of influenza a virus infection using a549 cell membrane components

This example demonstrates that at the in vitro cell level, the a549 cell membrane fraction is effective in inhibiting influenza a virus infection of target cells (wild-type a 549).

Two influenza a viruses were selected, type a/California/07/2009H1N 1(a/California/07/2009H1N1) and type a/shanghai/4664T H7N9(a/shanghai/4664T/2013H7N9) (saved in the third biosafety laboratory of the public health clinical centre of shanghai), and relevant experiments were performed in the second biosafety laboratory and the third biosafety laboratory, respectively.

100,000 wild-type A549 cells were plated 12 hours in advance in 24-well cell culture plates. 100. mu.l of a dilution (500. mu.g/ml, 250. mu.g/ml, 125. mu.g/ml) of the cell membrane fraction prepared in example 2 and 100. mu.l of a solution containing 100,000 infectious influenza virus particles (multiplicity of infection MOI of 1) were incubated at 37 ℃ for 1 hour, and then added to the cells prepared in advance, and a cell sample was taken 8 hours after infection. Expression levels of influenza Nucleoprotein (NP) and polymerase protein (PB2), as well as cytoskeletal protein (GAPDH), were detected using western blot experiments.

FIGS. 4a/b show the Western blot results of cell membrane fractions inhibiting infection with influenza A virus H1N1 and the relative inhibition of viral protein expression (expression levels in membrane-treated versus PBS-treated groups), respectively. FIGS. 4c/d are the Western blot results of cell membrane fractions inhibiting infection with influenza A virus H7N9 and the relative inhibition of viral protein expression (expression levels of membrane-treated versus PBS-treated).

The results show that the wild-type A549 cell membrane component can effectively inhibit the target cells A549 infected by the influenza A viruses H1N1 (figure 4a/b) and H7N9 (figure 4 c/d).

The results prove that the wild A549 cell membrane component has an effective inhibition effect on influenza virus infection.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shanghai city public health clinic center

<120> method for preventing and treating respiratory infectious diseases by using respiratory epithelial cell membrane and application thereof

<130> 204699 1CNCN

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 2421

<212> DNA

<213> Artificial sequence

<220>

<221> CDS

<222> (1)..(2421)

<400> 1

atg tca agc tct tcc tgg ctc ctt ctc agc ctt gtt gct gta act gct 48

Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala

1 5 10 15

gct cag tcc acc att gag gaa cag gcc aag aca ttt ttg gac aag ttt 96

Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe

20 25 30

aac cac gaa gcc gaa gac ctg ttc tat caa agt tca ctt gct tct tgg 144

Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp

35 40 45

aat tat aac acc aat att act gaa gag aat gtc caa aac atg aat aat 192

Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn

50 55 60

gct ggg gac aaa tgg tct gcc ttt tta aag gaa cag tcc aca ctt gcc 240

Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala

65 70 75 80

caa atg tat cca cta caa gaa att cag aat ctc aca gtc aag ctt cag 288

Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln

85 90 95

ctg cag gct ctt cag caa aat ggg tct tca gtg ctc tca gaa gac aag 336

Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys

100 105 110

agc aaa cgg ttg aac aca att cta aat aca atg agc acc atc tac agt 384

Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser

115 120 125

act gga aaa gtt tgt aac cca gat aat cca caa gaa tgc tta tta ctt 432

Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu

130 135 140

gaa cca ggt ttg aat gaa ata atg gca aac agt tta gac tac aat gag 480

Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu

145 150 155 160

agg ctc tgg gct tgg gaa agc tgg aga tct gag gtc ggc aag cag ctg 528

Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu

165 170 175

agg cca tta tat gaa gag tat gtg gtc ttg aaa aat gag atg gca aga 576

Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg

180 185 190

gca aat cat tat gag gac tat ggg gat tat tgg aga gga gac tat gaa 624

Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu

195 200 205

gta aat ggg gta gat ggc tat gac tac agc cgc ggc cag ttg att gaa 672

Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu

210 215 220

gat gtg gaa cat acc ttt gaa gag att aaa cca tta tat gaa cat ctt 720

Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu

225 230 235 240

cat gcc tat gtg agg gca aag ttg atg aat gcc tat cct tcc tat atc 768

His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile

245 250 255

agt cca att gga tgc ctc cct gct cat ttg ctt ggt gat atg tgg ggt 816

Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly

260 265 270

aga ttt tgg aca aat ctg tac tct ttg aca gtt ccc ttt gga cag aaa 864

Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys

275 280 285

cca aac ata gat gtt act gat gca atg gtg gac cag gcc tgg gat gca 912

Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala

290 295 300

cag aga ata ttc aag gag gcc gag aag ttc ttt gta tct gtt ggt ctt 960

Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu

305 310 315 320

cct aat atg act caa gga ttc tgg gaa aat tcc atg cta acg gac cca 1008

Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro

325 330 335

gga aat gtt cag aaa gca gtc tgc cat ccc aca gct tgg gac ctg ggg 1056

Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly

340 345 350

aag ggc gac ttc agg atc ctt atg tgc aca aag gtg aca atg gac gac 1104

Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp

355 360 365

ttc ctg aca gct cat cat gag atg ggg cat atc cag tat gat atg gca 1152

Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala

370 375 380

tat gct gca caa cct ttt ctg cta aga aat gga gct aat gaa gga ttc 1200

Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe

385 390 395 400

cat gaa gct gtt ggg gaa atc atg tca ctt tct gca gcc aca cct aag 1248

His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys

405 410 415

cat tta aaa tcc att ggt ctt ctg tca ccc gat ttt caa gaa gac aat 1296

His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn

420 425 430

gaa aca gaa ata aac ttc ctg ctc aaa caa gca ctc acg att gtt ggg 1344

Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly

435 440 445

act ctg cca ttt act tac atg tta gag aag tgg agg tgg atg gtc ttt 1392

Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe

450 455 460

aaa ggg gaa att ccc aaa gac cag tgg atg aaa aag tgg tgg gag atg 1440

Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met

465 470 475 480

aag cga gag ata gtt ggg gtg gtg gaa cct gtg ccc cat gat gaa aca 1488

Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr

485 490 495

tac tgt gac ccc gca tct ctg ttc cat gtt tct aat gat tac tca ttc 1536

Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe

500 505 510

att cga tat tac aca agg acc ctt tac caa ttc cag ttt caa gaa gca 1584

Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala

515 520 525

ctt tgt caa gca gct aaa cat gaa ggc cct ctg cac aaa tgt gac atc 1632

Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile

530 535 540

tca aac tct aca gaa gct gga cag aaa ctg ttc aat atg ctg agg ctt 1680

Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu

545 550 555 560

gga aaa tca gaa ccc tgg acc cta gca ttg gaa aat gtt gta gga gca 1728

Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala

565 570 575

aag aac atg aat gta agg cca ctg ctc aac tac ttt gag ccc tta ttt 1776

Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe

580 585 590

acc tgg ctg aaa gac cag aac aag aat tct ttt gtg gga tgg agt acc 1824

Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr

595 600 605

gac tgg agt cca tat gca gac caa agc atc aaa gtg agg ata agc cta 1872

Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu

610 615 620

aaa tca gct ctt gga gat aaa gca tat gaa tgg aac gac aat gaa atg 1920

Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met

625 630 635 640

tac ctg ttc cga tca tct gtt gca tat gct atg agg cag tac ttt tta 1968

Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu

645 650 655

aaa gta aaa aat cag atg att ctt ttt ggg gag gag gat gtg cga gtg 2016

Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val

660 665 670

gct aat ttg aaa cca aga atc tcc ttt aat ttc ttt gtc act gca cct 2064

Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro

675 680 685

aaa aat gtg tct gat atc att cct aga act gaa gtt gaa aag gcc atc 2112

Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile

690 695 700

agg atg tcc cgg agc cgt atc aat gat gct ttc cgt ctg aat gac aac 2160

Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn

705 710 715 720

agc cta gag ttt ctg ggg ata cag cca aca ctt gga cct cct aac cag 2208

Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln

725 730 735

ccc cct gtt tcc ata tgg ctg att gtt ttt gga gtt gtg atg gga gtg 2256

Pro Pro Val Ser Ile Trp Leu Ile Val Phe Gly Val Val Met Gly Val

740 745 750

ata gtg gtt ggc att gtc atc ctg atc ttc act ggg atc aga gat cgg 2304

Ile Val Val Gly Ile Val Ile Leu Ile Phe Thr Gly Ile Arg Asp Arg

755 760 765

aag aag aaa aat aaa gca aga agt gga gaa aat cct tat gcc tcc atc 2352

Lys Lys Lys Asn Lys Ala Arg Ser Gly Glu Asn Pro Tyr Ala Ser Ile

770 775 780

gat att agc aaa gga gaa aat aat cca gga ttc caa aac act gat gat 2400

Asp Ile Ser Lys Gly Glu Asn Asn Pro Gly Phe Gln Asn Thr Asp Asp

785 790 795 800

gtt cag acc tcc ttt tag tag 2421

Val Gln Thr Ser Phe

805

<210> 2

<211> 805

<212> PRT

<213> Artificial sequence

<400> 2

Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala

1 5 10 15

Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe

20 25 30

Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp

35 40 45

Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn

50 55 60

Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala

65 70 75 80

Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln

85 90 95

Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys

100 105 110

Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser

115 120 125

Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu

130 135 140

Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu

145 150 155 160

Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu

165 170 175

Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg

180 185 190

Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu

195 200 205

Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu

210 215 220

Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu

225 230 235 240

His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile

245 250 255

Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly

260 265 270

Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys

275 280 285

Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala

290 295 300

Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu

305 310 315 320

Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro

325 330 335

Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly

340 345 350

Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp

355 360 365

Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala

370 375 380

Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe

385 390 395 400

His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys

405 410 415

His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn

420 425 430

Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly

435 440 445

Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe

450 455 460

Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met

465 470 475 480

Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr

485 490 495

Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe

500 505 510

Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala

515 520 525

Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile

530 535 540

Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu

545 550 555 560

Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala

565 570 575

Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe

580 585 590

Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr

595 600 605

Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu

610 615 620

Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met

625 630 635 640

Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu

645 650 655

Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val

660 665 670

Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro

675 680 685

Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile

690 695 700

Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn

705 710 715 720

Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln

725 730 735

Pro Pro Val Ser Ile Trp Leu Ile Val Phe Gly Val Val Met Gly Val

740 745 750

Ile Val Val Gly Ile Val Ile Leu Ile Phe Thr Gly Ile Arg Asp Arg

755 760 765

Lys Lys Lys Asn Lys Ala Arg Ser Gly Glu Asn Pro Tyr Ala Ser Ile

770 775 780

Asp Ile Ser Lys Gly Glu Asn Asn Pro Gly Phe Gln Asn Thr Asp Asp

785 790 795 800

Val Gln Thr Ser Phe

805

<210> 3

<211> 805

<212> PRT

<213> Artificial sequence

<400> 3

Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala

1 5 10 15

Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe

20 25 30

Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp

35 40 45

Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn

50 55 60

Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala

65 70 75 80

Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln

85 90 95

Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys

100 105 110

Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser

115 120 125

Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu

130 135 140

Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu

145 150 155 160

Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu

165 170 175

Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg

180 185 190

Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu

195 200 205

Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu

210 215 220

Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu

225 230 235 240

His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile

245 250 255

Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly

260 265 270

Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys

275 280 285

Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala

290 295 300

Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu

305 310 315 320

Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro

325 330 335

Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly

340 345 350

Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp

355 360 365

Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala

370 375 380

Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe

385 390 395 400

His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys

405 410 415

His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn

420 425 430

Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly

435 440 445

Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe

450 455 460

Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met

465 470 475 480

Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr

485 490 495

Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe

500 505 510

Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala

515 520 525

Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile

530 535 540

Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu

545 550 555 560

Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala

565 570 575

Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe

580 585 590

Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr

595 600 605

Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu

610 615 620

Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met

625 630 635 640

Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu

645 650 655

Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val

660 665 670

Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro

675 680 685

Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile

690 695 700

Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn

705 710 715 720

Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln

725 730 735

Pro Pro Val Ser Ile Trp Leu Ile Val Phe Gly Val Val Met Gly Val

740 745 750

Ile Val Val Gly Ile Val Ile Leu Ile Phe Thr Gly Ile Arg Asp Arg

755 760 765

Lys Lys Lys Asn Lys Ala Arg Ser Gly Glu Asn Pro Tyr Ala Ser Ile

770 775 780

Asp Ile Ser Lys Gly Glu Asn Asn Pro Gly Phe Gln Asn Thr Asp Asp

785 790 795 800

Val Gln Thr Ser Phe

805

<210> 4

<211> 3825

<212> DNA

<213> Artificial sequence

<400> 4

atgttcgtgt tcctggtgct cctcccactg gtgtcttctc agtgcgtgaa cctgaccacc 60

cggacacagc tgcccccagc ctacacaaac tctttcacac ggggcgtgta ctaccccgac 120

aaggtgttcc ggtctagcgt gctgcactct acccaggacc tgttcctccc attcttctct 180

aacgtgacat ggttccacgc catccacgta tccggcacaa acggcacaaa gcgcttcgac 240

aaccccgtgc tccctttcaa cgacggcgtg tacttcgcca gcaccgagaa gtctaacatt 300

atccggggct ggattttcgg caccacactg gactctaaga cccagtctct cctgattgtg 360

aacaacgcca ccaacgtggt gattaaggtg tgcgagttcc agttctgcaa cgaccctttc 420

ctgggcgtgt actaccacaa gaacaacaag tcttggatgg agtctgagtt ccgggtgtac 480

tctagcgcca acaactgcac cttcgagtac gtgtctcagc ctttcctcat ggacttggaa 540

ggcaagcagg gcaacttcaa gaacctgcgg gagttcgtgt tcaagaacat tgacggctac 600

ttcaagattt actctaagca cacccctatt aacctcgtga gggacctccc tcagggcttc 660

tccgcccttg agccactggt ggacctccca attggcatta acatcacacg gttccagaca 720

ctcctcgccc tccaccgaag ttacctgacc cccggcgact ctagctctgg ctggacagcc 780

ggcgccgccg cctactacgt gggctacctg caaccacgca cattcctcct gaagtacaac 840

gagaacggca caattaccga cgccgtggac tgcgccctcg acccactgag cgagacaaag 900

tgcacactga agtctttcac agtggagaag ggcatttacc agacatctaa cttcagagtg 960

cagcctacag agtctattgt gcggttccca aacattacaa acctgtgccc tttcggcgag 1020

gtgttcaacg ccacccgctt cgcctctgtg tacgcctgga accggaagcg gatttctaac 1080

tgcgtggccg actactctgt gctgtacaac tccgcctctt tctctacatt caagtgctac 1140

ggcgtgtctc ctacaaagct gaacgacctg tgcttcacca acgtgtacgc cgacagcttc 1200

gtgatcaggg gcgacgaggt gaggcagatc gccccaggcc agacaggcaa gattgccgac 1260

tacaactaca agctccctga cgacttcaca ggctgcgtga ttgcctggaa ctctaacaac 1320

ctcgactcta aggtgggcgg caactacaac tacctgtaca ggctgttccg gaagtccaac 1380

ctgaagcctt tcgagaggga cattagcacc gagatttacc aggccggcag caccccatgc 1440

aacggcgtgg agggcttcaa ctgctacttc cccctacaat cttacggctt ccagcctaca 1500

aacggcgtgg gctaccagcc ataccgggtg gtggtgctgt ccttcgagct gctccacgcc 1560

cccgccacag tgtgcggccc aaagaagagc acaaacctcg tgaagaacaa gtgcgtgaac 1620

ttcaacttca acggcctgac aggcacaggc gtgctcacag agtctaacaa gaagttcctg 1680

ccattccagc agttcggacg ggacattgcc gacaccaccg acgccgtgag ggaccctcag 1740

acactggaga ttctcgacat caccccatgc agcttcggcg gcgtgtccgt gatcacccca 1800

ggcacaaaca catctaacca ggtggccgtg ctgtaccagg acgtgaactg caccgaggtg 1860

ccagtggcca tccacgccga ccagctcacc ccaacatgga gagtgtactc cacaggctct 1920

aacgtgttcc agacacgcgc cggctgcctg attggcgccg agcatgtcaa caactcttac 1980

gagtgcgaca tccctattgg cgccggcatt tgcgcctctt accagaccca gacaaacagc 2040

cctagacggg cccggtctgt ggcctctcag tctattattg cctacaccat gtctctgggc 2100

gccgagaact ctgtggccta ctctaacaac tctattgcca tccctacaaa cttcacaatt 2160

tctgtgacca ccgagatcct ccccgtgagc atgactaaga catcagttga ctgcaccatg 2220

tacatttgcg gcgactccac cgagtgctct aacctcctcc tgcaatacgg ctctttctgc 2280

acacagctca accgggccct cacaggcatc gccgtggagc aggacaagaa cacccaggag 2340

gtgttcgccc aggtgaagca gatttacaag acccctccta ttaaggactt cggcggcttc 2400

aacttctctc agattctccc cgaccctagc aagccatcta agcggtcttt cattgaggac 2460

ctcctgttca acaaggtgac actggccgac gccggcttca ttaagcagta cggcgactgc 2520

ctgggcgaca ttgccgcacg agacctgatt tgcgcccaga agttcaacgg cctcacagtg 2580

ctccccccac tgctcaccga cgagatgatc gcccagtaca catccgccct gctggccggc 2640

acaattacat ctggctggac attcggcgcc ggcgccgccc tccaaatccc tttcgccatg 2700

cagatggcct accggttcaa cggcattggc gtgacccaga acgtgctgta cgagaaccag 2760

aagctcatcg ccaaccagtt caacagcgcc atcggcaaga ttcaggactc tctgtctagc 2820

acagccagcg ccctgggcaa gctccaggac gtggtgaacc agaacgccca ggccctcaac 2880

acactggtga agcagctgtc tagcaacttc ggcgccatta gctctgtgct gaacgacatt 2940

ctgagtcgtc tcgacaaggt ggaggccgag gtgcagattg acaggctcat cacaggcaga 3000

cttcaatcct tgcagacata cgtgacccag cagctgatta gagccgccga gattagagcg 3060

agtgctaatt tagccgccac aaagatgagc gagtgcgtgc tgggccagtc caagcgggtg 3120

gacttctgcg gcaagggcta ccacctcatg tctttccctc agtccgcccc tcacggcgtg 3180

gtgttcctcc acgtaacata cgtgcccgcc caggagaaga acttcaccac agcccccgcc 3240

atttgccacg acggcaaggc ccacttccct agagagggcg tgttcgtgtc taacggcacc 3300

cactggttcg tgacccagcg gaacttctac gagcctcaga tcatcaccac cgacaacacc 3360

ttcgtgtccg gcaactgcga cgtggtgatt ggcattgtga acaacacagt gtacgaccca 3420

cttcagcctg aactcgactc tttcaaggag gaactagaca agtacttcaa gaaccacaca 3480

tctcccgacg tggacctggg cgacattagc ggcattaacg cctctgtggt gaacattcag 3540

aaggagattg acagactgaa cgaggtggcc aagaacctga acgagagcct gatcgacctc 3600

caggagctgg gcaagtacga gcagtacatt aagtggcctt ggtacatttg gctgggcttc 3660

attgccggcc tgattgccat tgtgatggtg acaattatgc tgtgctgcat gacatcttgc 3720

tgctcttgcc tgaagggctg ctgctcttgc ggctcttgct gcaagttcga cgaggacgac 3780

tctgagcccg tgctgaaggg cgtgaagctc cactacacct gatga 3825

Claims (10)

1. Use of a cell membrane of an epithelial cell of the respiratory tract for the preparation of a product for the prevention and/or treatment of infection by a pathogen of an infectious respiratory disease.

2. Use according to claim 1, wherein the respiratory epithelial cells are selected from: lung epithelial cells, tracheal epithelial cells, bronchial epithelial cells, nasopharyngeal epithelial cells, for example said epithelial cells are selected from A549 cells, Calu-1 cells, Calu-3 cells, BEAS-2B cells or NP69 cells.

3. Use according to claim 1, wherein the cell membrane is an unmodified cell membrane or a modified cell membrane, e.g. the cell membrane is a cell membrane of a wild-type respiratory epithelial cell, a genetically engineered cell membrane, a chemically modified cell membrane.

4. The use of claim 3, wherein the modified cell membrane is selected from the group consisting of: a cell membrane modified to have overexpressed respiratory infectious disease pathogen cell surface receptors, immunomodulatory molecules, and/or antipathogenic molecules thereon; a cell membrane modified to express thereon a respiratory infectious disease pathogen cell surface receptor, immunomodulatory molecule, and/or antipathogenic molecule not present in wild type; or any combination of the above.

5. The use according to claim 3, wherein,

the cell surface receptor is one or more selected from the group consisting of: sialic acid or sialic acid modified glycoproteins, angiotensin converting enzyme 2(ACE2), CD26 molecules, CD13 peptidase (APN), CD46, SLAM family molecules, CX3CR1, and insulin-like growth factor (IGF-2);

and/or

The immunoregulatory molecule or antipathogenic molecule is one or more selected from the group consisting of: IFITM1, IFN-k, IL-7, IL-15, IL-21, IL-6 receptor, TNF-a receptor, IL-1 receptor, class I interferon receptor or subunit thereof and toxin receptor.

6. The use of claim 1, wherein the respiratory infectious disease agent is one or more selected from the group consisting of: influenza viruses (e.g., H1N1, H3N2, H7N9, H5N1, etc.), coronaviruses (e.g., SARS-CoV-2, MERS-CoV), mumps virus, measles virus, respiratory syncytial virus, Nipah virus, human metapneumovirus, and Chlamydia pneumoniae.

7. Use according to claim 1, wherein the product is a medicament.

8. A method for preparing a cell membrane and/or a product as mentioned in any of claims 1 to 7, comprising:

(a) providing airway epithelial cells, wherein said cells are modified or unmodified; and

(b) isolating the cell membrane of the cell to obtain an unmodified cell membrane or a modified cell membrane;

(c) optionally, the separated cell membrane is manufactured into a product, such as directly into a drug, coated or coupled to a carrier material (e.g., a biodegradable nanomaterial) to form a drug (e.g., a nanoparticle drug).

9. The method of claim 8, wherein the cells are engineered to overexpress a cell surface receptor, immunomodulatory molecule, and/or antipathogenic molecule of a respiratory infectious disease pathogen or to express a cell surface receptor, immunomodulatory molecule, and/or antipathogenic molecule of a respiratory infectious disease pathogen not present in wild-type.

10. A product comprising a cell membrane of an airway epithelial cell as referred to in any one of claims 1 to 7 or comprising a cell membrane prepared by the method of claim 8 or 9.

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