CN118086212A - Dendritic cell sensitized by novel coronavirus epitope polypeptide and application thereof - Google Patents

Abstract

The invention provides dendritic cells sensitized by novel coronavirus epitope polypeptides and application thereof. Specifically provided is a dendritic cell targeting novel coronavirus (SARS-CoV-2) M protein, which is sensitized by novel coronavirus M protein restriction epitope polypeptide, and can induce the generation of the epitope polypeptide specific cytotoxicity T cell. Also provides a preparation method and application of the dendritic cell.

Description

Dendritic cell sensitized by novel coronavirus epitope polypeptide and application thereof

Technical Field

The present invention relates to the field of immunology and medicine, more specifically to dendritic cells sensitized by a novel coronavirus SARS-CoV-2HLA-A2 restriction epitope polypeptide (e.g. polypeptide of SEQ ID NO: 5), compositions comprising said sensitized dendritic cells and uses thereof, e.g. for the preparation of detection, diagnosis, prevention and/or treatment products for SARS-CoV-2 related diseases.

Background

Dendritic cells (DENDRITIC CELL, DC) are a class of immune cells found by the Ralph M.Steinman professor of the 2011 Nobel physiology or medicine prize, and are known for their mature morphology to protrude through many dendritic-like or pseudopodiform projections. DCs have a key role in the acquired immune response and are the strongest professional antigen presenting cells (ANTIGEN PRESENTING CELLS, APC) known at present, which can efficiently ingest, process, handle and present antigens, mature DCs are the most powerful antigen presenting cells currently known in humans, which can only activate T cells in resting state, can activate cd4+ helper T cells (T helper lymphocyte, th) and cytotoxic T Cells (CTLs), and are central to the initiation, regulation and maintenance of immune responses.

DC is widely distributed in various parts of human body, such as blood, liver, spleen, lymph node, lung, kidney, gastrointestinal tract and other tissue interstitials, and accounts for about 0.5-1% of the total number of peripheral blood mononuclear cells. The classification of the DC subpopulations is complex, bone marrow-derived DC (mDC) and lymphoid-derived DC (pDC) are two major types of peripheral blood DCs. DCs can be generated via monocyte differentiation, and monocytes in PBMC isolated for in vitro culture, GM-CSF and IL-4 induced to generate immature DCs. Immature DCs can differentiate into mature cells through different maturation-inducing components, and the maturation state of the DCs is a critical factor in determining the effectiveness of DC vaccines. Immature DCs have limited ability to sensitize T cells and may also induce T cell tolerance. In addition, the mature DC has stronger migration capability and can more effectively migrate to the T cell area of the secondary lymphoid tissue so as to induce immune response.

DC vaccine, i.e. antigen sensitized DC cells such as antigen protein/polypeptide, uses the powerful antigen presenting function to activate immune response such as T cells in the patient, thereby playing a therapeutic role, and belongs to a therapeutic vaccine. The main mechanism of therapeutic effect of DC vaccine is mainly through the following three pathways: (1) DC ingests antigen information, then processes and presents to CD4+ T cells to activate and exert therapeutic effects; or presented to cd8+ T cells to activate them to produce specific cytotoxic T cells that exert therapeutic effects against the particular pathogen; (2) DCs present more antigenic peptides by increasing the expression level of cell surface histocompatibility complex (MHC) class I, II molecules, allowing the corresponding T Cell Receptor (TCR) to be fully bound; at the same time, DC provides high levels of B7-1 (CD 80), B7-2 (CD 86), CD40, etc. co-stimulatory molecules, allowing T cells to be fully activated; (3) DCs act synergistically by synthesizing and secreting some cytokines, such as IL-12.IL-12 induces T cells, NK cells to produce large amounts of tumor necrosis factor, perforin and granzyme, thereby dissolving tumor cells and the like.

However, there is still a need for further research and development of the role of dendritic cells in the prevention and/or treatment of other diseases other than tumors, in particular viral infectious diseases.

To date, three highly pathogenic human coronaviruses (CoVs) have been identified, including the middle east respiratory syndrome coronavirus (MERS-CoV), the Severe Acute Respiratory Syndrome (SARS) coronavirus (SARS-CoV), and a 2019 new coronavirus (SARS-CoV-2, abbreviated new coronavirus).

The main clinical symptoms of patients infected by the novel coronavirus are fever, cough, shortness of breath and the like, and the laboratory examination frequently shows multiple grinding glass shadows of the lung. Part of the patient's condition may rapidly deteriorate and serious complications occur, including acute respiratory distress syndrome, acute kidney injury, secondary infections, inflammatory factor storms, etc., and part of the patient eventually dies from respiratory failure, multiple organ dysfunction or shock.

The pathogen SARS-CoV-2 causing disease is identified by laboratory as a newly discovered beta genus coronavirus, which has 79% genetic similarity with the pathogen SARS-CoV genus, has a diameter of 60-140 nm, and has an envelope and a single-stranded, sense RNA genome. The SARS-CoV-2 genome is flanked by 5' and 3' untranslated regions, the 5' end comprising 2 longer Open Reading Frames (ORFs), encoding 16 nonstructural proteins; the remaining near 3' end of the genome encodes mainly structural proteins and other accessory proteins. The structural proteins of the virus mainly include spike protein (S protein), membrane glycoprotein (M protein), small envelope protein (envelope glycoprotein, E protein), nucleocapsid protein (nucleocapsid protein, N protein).

The S protein plays the most important role in the attachment, fusion and entry processes of viruses and is also the main target of antibodies, entry inhibitors and vaccines. The S protein mediates entry of the virus into the host cell, first through the Receptor Binding Domain (RBD) of the S1 subunit to bind to the host receptor, and then through the S2 subunit to fuse the virus and the host cell membrane. The S protein can bind host cells and mediate virus infection, is the main antigen protein for reference in the research and development of antibodies and vaccines at present, but SARS-CoV-2 continuously accumulates mutation, especially mutation of structural protein, in the host, so that a plurality of SARS-CoV-2 mutant strains with adaptability advantages, such as amikacin and the like, are produced. These mutants generally have a stronger infectious or pathogenic potential and mutations may lead to alterations in antigenic properties, thereby affecting the control of prophylactic vaccines and therapeutic antibodies against the mutants, resulting in immune escape of the virus in the body.

M protein is essentially a kind of transmembrane protein, and is structurally characterized by having three structural domains, namely an N-terminal extracellular domain, a three-transmembrane domain and an internal C-terminal domain, and plays an important role in the morphogenesis and maintenance of viruses, and is the most abundant glycoprotein in virus particles. More critical is that the M gene is relatively conserved, the frequency of mutation of the M protein is far lower than that of the S protein, and vaccines or antibodies developed based on the M protein are not easy to cause reduction of prevention and treatment effects due to virus mutation. Therefore, if the M protein is used as a target for immunization of an organism, on one hand, the organism can generate specific immune response aiming at the virus M protein, so that the organism can effectively remove viruses, measures are provided for preventing and treating SARS-CoV-2, and on the other hand, the relatively conservative low mutation characteristic can further avoid immune escape of the viruses caused by mutation, and the immune escape has a broader-spectrum effect.

There remains a great need in the art to develop effective medicaments and methods for the effective prevention and treatment of the novel coronavirus SARS-CoV-2 and its related diseases.

Disclosure of Invention

The present application provides sensitized dendritic cells and related products and products thereof, which can be directly or indirectly applied to detection, diagnosis, prevention and/or treatment of novel coronavirus related diseases.

In some aspects herein, a dendritic cell targeting a novel coronavirus (SARS-CoV-2) M protein is provided.

In some embodiments, the dendritic cells herein are sensitized by SARS-CoV-2M protein epitope polypeptide.

In some embodiments, the epitope polypeptide for sensitizing dendritic cells comprises the amino acid sequence: FLWLLWPVT (SEQ ID NO: 5). In some embodiments, the epitope polypeptide used to sensitize the dendritic cell has an amino acid sequence FLWLLWPVT (SEQ ID NO: 5).

In some embodiments, the epitope polypeptide used to sensitize the dendritic cell is an HLA-A2 restriction epitope peptide.

In some embodiments, the epitope polypeptide used to sensitize the dendritic cell has an affinity coefficient for the cell surface HLA-A x 0201 molecule of at least 2.0, e.g., at least 2.2, at least 2.5, at least 2.9.

In some embodiments, the dendritic cells herein are mature dendritic cells, e.g., express surface molecules characteristic of mature dendritic cells (e.g., CD80, CD83, CD 86).

In some embodiments, the dendritic cell surface herein presents a SARS-CoV-2M protein antigen.

In some embodiments, the sensitized dendritic cells herein have enhanced antigen presentation capacity compared to a non-sensitized reference dendritic cell.

In some embodiments, the dendritic cells herein are derived from bone marrow cells, umbilical cord blood cells, peripheral blood mononuclear cells.

In some embodiments, the dendritic cells herein are derived from a mammal, e.g., human, non-human primate, murine.

In some aspects, an article of manufacture is provided comprising a dendritic cell herein.

In some embodiments, the preparation is a cellular preparation.

In some embodiments, the form of the dendritic cells in the preparation may be selected from the group consisting of: dendritic cells alone or in combination with viral therapeutic agents, cytotoxic agents, radionuclides, toxins, detectable labels, prodrug activating enzymes.

In some aspects, there is provided the use of a dendritic cell and/or article of manufacture herein in the manufacture of a product for the detection, diagnosis, prevention and/or treatment of a SARS-CoV-2 related disease or disorder.

In some embodiments, the product is selected from: dendritic cell vaccines, adoptive cell therapy drugs, effector cell stimulators, or combinations thereof. In some embodiments, the product comprises a primed dendritic cell of the application and/or a specific T cell induced with said primed dendritic cell.

In some aspects, methods of detecting, diagnosing, preventing and/or treating a SARS-CoV-2 associated disease or condition are provided, the methods comprising directly or indirectly detecting, diagnosing, preventing and/or treating a SARS-CoV-2 associated disease or condition using a dendritic cell or preparation as described herein.

In some embodiments, methods of preventing and/or treating a SARS-CoV-2 associated disease or disorder are provided, comprising administering to a subject in need thereof a dendritic cell or preparation as described herein or an activated effector cell prepared using the dendritic cell or preparation.

In some aspects, there is provided a method of preparing a dendritic cell described herein, the method comprising:

(a) Providing SARS-CoV-2M protein epitope polypeptide;

(b) Sensitizing dendritic cells or precursors thereof with the epitope polypeptides.

In some embodiments, the SARS-CoV-2M protein epitope polypeptide used in the method of making the dendritic cell comprises a sequence of WLLWPV. In some embodiments, the epitope polypeptide used to sensitize the dendritic cell has an amino acid sequence FLWLLWPVT (SEQ ID NO: 5). In some embodiments, the epitope polypeptide used to sensitize the dendritic cell is an HLA-A2 restriction epitope peptide.

Any combination of the technical solutions and features described above can be made by a person skilled in the art without departing from the inventive concept and the scope of protection 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 be further described with reference to the accompanying drawings, wherein these drawings are provided only for illustrating embodiments of the present invention and are not intended to limit the scope of the present invention.

Fig. 1: SMp-11 epitope polypeptides have high affinity to HLA-A x 0201 molecules. Table 1 shows the results of a flow assay for predicting binding of an epitope peptide to HLA-A 0201 molecules, with higher fluorescence coefficients representing higher affinity of the epitope peptide to HLA-A 0201 (generally, higher fluorescence coefficients than 1 represent high affinity of the epitope peptide to HLA-A 0201).

Fig. 2: the murine DC immunized transgenic mice sensitized with the epitope peptide induced a specific immune response against the epitope. The figure shows analysis of Elispot results (", P <0.0001," ns "= no significant difference).

Fig. 3: mature SMp-11 epitope polypeptide sensitized human Dendritic Cells (DCs) were successfully induced in vitro. The figure shows the expression strength of DC cell surface maturation marker molecules after induced culture by flow detection SMp-11.

Fig. 4: SMp-11 sensitized human DC induced effector T lymphocytes have SMp-11 epitope specific killing effect. The graph shows that CFSE/7-AAD flow assay analysis induced SMp-11 specific CTL killing (", P < 0.05"; "P <0.0001," ns "= no significant difference).

Fig. 5: SMp-11 specific human CTL can specifically secrete killer cell factor IFN-gamma under the stimulation of target cells. The figure shows analysis of Elispot results (", P <0.0001," ns "= no significant difference) (target cells loaded with irrelevant peptide OVA as control group).

Detailed Description

The application provides dendritic cells sensitized by SARS-CoV-2M protein epitope polypeptide and products thereof, wherein the dendritic cells have characteristic molecules of mature dendritic cells, have stronger antigen presenting capability, and can efficiently induce the generation and activation of effector cells, thereby having great practical value in the detection, diagnosis, prevention and/or treatment of novel coronavirus related diseases.

Specifically, we sensitized dendritic cell precursors (such as peripheral blood mononuclear cells) from various sources by using HLA-A2 restriction epitope polypeptide of SARS-CoV-2 novel coronavirus M protein obtained by screening to obtain mature sensitized dendritic cells, and the dendritic cells can highly express mature dendritic cell characteristic molecules such as CD80, CD83, CD86 and the like. Further studies have also found that sensitized dendritic cells are able to efficiently induce specific, HLA-A 0201-restricted cytotoxic T lymphocytes. Thus, the application has important significance for the development of SARS-CoV-2 preventive and/or therapeutic vaccine and preparation.

The features mentioned in the description or the features mentioned in the examples can be combined. All of the features disclosed in this specification may be combined with any combination of the features disclosed in this specification, and the various features disclosed in this specification may be substituted for any alternative feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the disclosed features are merely general examples of equivalent or similar features.

All numerical ranges provided herein are intended to expressly include all values and ranges of values between the endpoints of the range. For example, 1-3 includes endpoints 1 and 3, specific integer number points 2 and non-integer number points therein (e.g., without limitation, 1.2, 1.5, 1.8, 2.1, 2.3, 2.4, 2.8, etc.), and sub-ranges thereof (e.g., without limitation, 1-2, 2-3, 1-1.2, 1.5-1.8, etc.).

As used herein, "comprising," having, "or" including "includes" including, "" consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … …; "consisting essentially of … …", "consisting essentially of … …" and "consisting of … …" are under the notion of "containing", "having" or "including".

As used herein, "epitope polypeptide," "specific (poly) peptide," "HLA-A 2-restricted epitope (poly) peptide," and "SARS-CoV-2HLA-A 2-restricted epitope (poly) peptide" are used interchangeably to refer to an epitope peptide derived from the SARS-CoV-2-based M protein that has high affinity for HLA-A2 and is useful for sensitizing dendritic cells.

The epitope polypeptide used to sensitize the dendritic cells may include or be the polypeptide of SEQ ID NO. 5. The proteins or polypeptides of the invention may be naturally purified products, or chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher animal, insect, and mammalian cells) using recombinant techniques.

As used herein, the terms "Dendritic Cells (DCs) of the present invention" and "sensitized dendritic cells" are used interchangeably and refer to dendritic cells that are sensitized after stimulation with SARS-CoV-2M protein epitope polypeptides.

The dendritic cells used in the present invention may be obtained from mammals, preferably from rats, mice or humans. In one embodiment of the invention, the dendritic cells are obtained from a human. In another embodiment of the invention, the dendritic cells are obtained from a mouse.

The dendritic cells used in the preparation of the sensitized dendritic cells of the present invention can be immature dendritic cells or dendritic cell precursors. For example, dendritic cells can be induced from bone marrow cells, cord blood cells, peripheral blood mononuclear cells using methods known in the art (e.g., GM-CSF, IL-4, etc.).

In the present invention, the resulting dendritic cells are treated with an epitope polypeptide to obtain the dendritic cells of the present invention, the method comprising the steps of:

(1) Culturing the immature dendritic cells or precursors thereof in the presence of the epitope polypeptide and promoting maturation thereof;

(2) Collecting the treated dendritic cells.

In some embodiments, promoting maturation of the immature dendritic cells or precursors thereof comprises further adding a maturation stimulus, such as TNF- α, with the addition of an epitope polypeptide.

After collecting the dendritic cells of the present invention, the surface of the mature dendritic cells can be examined for their characteristic molecules (e.g., CD80, CD83, CD 86) and/or their specific antigen expression levels by flow cytometry or the like. In a preferred embodiment of the invention, the surface expression level of the sensitized dendritic cells of the invention is higher than the expression level of the mature dendritic cell-characteristic molecules of the dendritic cells prior to treatment.

The sensitized dendritic cells of the present invention promote phagocytic function of immature and mature dendritic cells, enhance antigen presenting function of dendritic cells, and promote effector cell production, and thus can be used for detecting, diagnosing, preventing and/or treating a novel coronavirus infectious disease or disorder.

The dendritic cells and/or articles herein can be used to prepare a variety of products. The product may include, but is not limited to, one or more selected from the group consisting of: a medicament, pharmaceutical composition or kit comprising an effective amount of the dendritic cells of the present invention and a pharmaceutically or immunologically acceptable carrier.

In preferred embodiments, the pharmaceutical compositions are useful for detecting, diagnosing, preventing and/or treating a disease associated with SARS-CoV-2, chronic diseases caused thereby, and/or conditions thereof. For example, the pharmaceutical composition of the present invention can be used for preventing or treating infectious diseases or symptoms caused by novel coronaviruses, such as lung or other tissue injuries, complications, multi-organ failure, etc. caused by the novel coronaviruses.

In some embodiments, the products herein may be used to prevent, eliminate, or reduce a novel coronavirus infection or at least one symptom thereof in a subject, such as respiratory symptoms (e.g., nasal obstruction, sore throat, hoarseness), headache, cough, sputum, fever, wheezing, dyspnea, pneumonia due to infection, severe acute respiratory syndrome, renal failure, and the like.

As used herein, "dendritic cell vaccine", "vaccine of the invention", "vaccine", "prophylactic or therapeutic vaccine" are used interchangeably and refer to a prophylactic and/or therapeutic vaccine prepared from the dendritic cells of the invention that targets the novel coronavirus of SARS-CoV-2, in particular its M protein.

The dendritic cells of the present invention can be directly administered to a subject in need of prevention or treatment by injection or the like. The in vitro treated dendritic cells or induced effector cells thereof may also be returned to the body of the cell supplier. The amount of cells is preferably 10 ⁴-10⁸ dendritic cells or effector cells/time, more preferably 10 ⁵-10⁷ dendritic cells or effector cells/time, most preferably 10 ⁶-10⁷ dendritic cells or effector cells/time.

The composition of the present invention may further comprise T lymphocytes or natural killer cells induced from spleen and peripheral blood. T lymphocytes and natural killer cells may also be administered prior to, at the time of, or after administration of the dendritic cells of the present invention.

As used herein, the term "comprising" or "including" includes "comprising," consisting essentially of … …, "and" consisting of … …. As used herein, the term "acceptable" ingredients are substances that are suitable for use in humans and/or animals and/or other subjects (e.g., cells) without undue adverse reactions (e.g., toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio. As used herein, the term "effective amount" refers to an amount that is functional or active in humans and/or animals and/or other subjects (e.g., cells) and acceptable to the subject.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, and may include various excipients and diluents. The term refers to such agent carriers: they are not per se essential active ingredients and are not overly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art and a sufficient discussion of pharmaceutically acceptable excipients can be found in Remington pharmaceutical sciences (Remington's Pharmaceutical Sciences, mack Pub.Co., N.J.1991).

Acceptable carriers in the compositions can contain liquids such as water, saline, glycerol, and ethanol. In addition, auxiliary substances such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting or emulsifying agents, flavoring agents, pH buffering substances, etc. may also be present in these carriers. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8.

The active substances in the composition of the invention account for 0.001 to 99.9 weight percent of the total weight of the composition; preferably 1 to 95wt%, more preferably 5 to 90wt%, and even more preferably 10 to 80wt% of the total weight of the composition. The rest is pharmaceutically acceptable carrier and other additives.

As used herein, the term "unit dosage form" refers to a dosage form that is required to prepare a composition of the present invention for administration in a single administration, including but not limited to various solid (e.g., tablet), liquid, capsule, sustained release formulations.

In another preferred embodiment of the invention, the composition is in unit dosage form or multiple dosage form. In another preferred embodiment of the invention, 1 to 6 doses of the composition of the invention, preferably 1 to 3 doses, are administered daily; most preferably, the daily dosage is 1 dose.

It will be appreciated that the effective dose of the active agent used may vary with the severity of the subject to be administered or treated. The specific conditions are determined according to the individual condition of the subject (e.g., the subject's weight, age, physical condition, effect to be achieved), which is within the scope of judgment of a skilled physician.

The composition of the invention can be solid (such as granules, tablets, freeze-dried powder, suppositories, capsules, sublingual tablets) or liquid (such as oral liquid) or other suitable shapes. In some embodiments, the compositions of the present invention may be administered by injection, such as subcutaneous injection, intravenous injection, infusion, or the like. In some embodiments, the route of administration may employ: (1) direct naked protein injection method; (2) Attaching an active substance to the transferrin/poly-L-lysine complex to enhance its biological effect; (3) Forming a complex of the active substance and positively charged lipid to overcome the difficulty of crossing cell membranes caused by negative charge of the phosphate backbone; (4) liposome-entrapped administration; (5) Binding to cholesterol increases its cytoplasmic retention time by a factor of 10; (6) Specific transport to target tissue and target cells using immunoliposome transport; (7) in vitro transfection; (8) electroporation (electroporation) into target cells.

The dendritic cells and related products and products thereof can be used in combination with antiviral infection medicines, and can also be used in combination with other medicines and treatment means for preventing and treating new coronavirus infectious diseases or symptoms.

Examples

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Appropriate modifications and variations of the invention may be made by those skilled in the art, and are within the scope of the invention.

The experimental methods described in the following examples, in which specific conditions are not specified, may be employed by conventional methods in the art, for example, with reference to the "molecular cloning Experimental guidelines" (third edition, new York, cold spring harbor laboratory Press, new York: cold Spring Harbor Laboratory Press, 1989) or according to the conditions recommended by the supplier. Methods for sequencing DNA are routine in the art and can also be provided for testing by commercial companies.

Percentages and parts are by weight unless otherwise indicated. 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 methods and materials described herein are presented for illustrative purposes only.

Example 1: affinity identification of epitope peptides with HLA-A x 0201 molecules

Peptide binding experiments were used to screen epitope peptides with high affinity to HLA-A x 0201. T2 cells (rich biotechnology, FH 0150) were first collected, washed three times with serum-free 1640 medium, and cell concentration was adjusted to 2×10 ⁶ cells/ml, plated in 24 well plates, 1 ml/well. Then incubated with 50. Mu.M of the candidate polypeptide and 3. Mu.g/ml of beta.2 microglobulin in a 5% CO ₂ incubator at 37℃for 18h. The incubated cells were washed three times with ice PBS, PE-labeled HLA-A 2-specific flow antibody (Biolegend Inc) was added, incubated at 4℃for 30min, and the average fluorescence intensity was measured by flow cytometry after PBS washing. The HLA-A2 restrictive influenza virus epitope polypeptide GILGFVFTL is used as a positive control, and the non-peptide stimulated simple T2 cells are used as a background control.

And (3) result judgment: the binding condition of the peptide and HLA-A 0201 molecule is detected by flow cytometry, and is based on that the binding of exogenous polypeptide and the MHC class I molecule on the surface of T2 cells can increase the expression quantity of the MHC class I molecule on the surface, and the more stable the binding of the exogenous polypeptide and the MHC class I molecule, the more the expression quantity of the MHC class I molecule can be detected, and the average fluorescence intensity is taken as a detection index. The result is a fluorescence coefficient (FI) as a measure. FI >1 of the polypeptide is considered to be a high affinity epitope.

The fluorescence coefficient (FI) is calculated as follows:

according to this method, the high affinity epitope polypeptide of HLA-A2 is selected from the membrane glycoprotein M protein (SEQ ID NO: 10), spike glycoprotein S protein (SEQ ID NO: 11), envelope protein E protein (SEQ ID NO: 12) of SARS-CoV-2 coronavirus. The average fluorescence intensity and fluorescence coefficient of exemplary epitope peptides involved in the screening can be seen in table 1 and fig. 1:

TABLE 1 fluorescence coefficient FI after flow detection of binding of different epitope peptides to T2 cell surface HLA-A 0201 molecules

Results: the affinity results of exemplary candidate epitope polypeptides with HLA-A x 0201 molecules are shown in table 1, and the results show that polypeptide epitopes No. 1, 3,5, 7, 8 (respectively named epitope peptide 1, epitope peptide 3, epitope peptide 5 (also called SMp-11), epitope peptide 7, and epitope peptide 8) are ranked in front, the average fluorescence intensities are all greater than 10000, the fluorescence coefficients FI are all greater than 1, and even all above 2.0; among them, epitope peptide 5 (also called SMp-11) FLWLLWPVT has a FI up to 2.73.

Conclusion: polypeptide epitopes with high affinity with HLA-A x 0201 molecules are obtained through screening, and the polypeptide epitopes (1, 3, 5, 7 and 8) with the top five ranks are selected for further screening and identification.

Example 2: IFN-gamma secretion detection of epitope peptide sensitized DC immune HLA-A2 transgenic mouse spleen cells

The resulting 5 potential polypeptide epitopes (epitope peptides 1,3, 5,7, 8) were screened according to example 1 and further tested for immunogenicity in HLA-A2 transgenic mice. Bone marrow derived Dendritic Cells (DCs) were prepared from HLA-A2.1/K ^b transgenic mice (Jackson Laboratory, 003475) by conventional methods. After the mice were neck-removed and sacrificed, the femur and tibia were removed, PBS was withdrawn with a 1ml syringe and a needle was inserted into the bone marrow cavity to flush out bone marrow, then bone marrow erythrocytes were lysed using Tris-NH ₄ Cl solution, the supernatant was discarded by centrifugation and DC were induced in 1640 medium containing 10% FBS, 1ng/ml mouse IL-4 (PeproTech, 214-14-5 UG), 10ng/ml mouse GM-CSF (PeproTech, 315-03-50 UG). Collecting DC cultured until 5 days, regulating cell concentration to 1× ⁶/ml, adding epitope peptide (final concentration 20 μg/ml), continuously culturing at 37deg.C in 5% CO ₂ incubator until 6 days, adding TNF-alpha (30 ng/ml) to stimulate maturation, and culturing until eighth day to obtain epitope peptide sensitized DC cell.

Peptide-sensitized DCs were collected, washed three times with PBS and cell concentrations were adjusted to 1X 10 ⁷/ml. Each male transgenic mouse was subcutaneously injected into the abdomen with 0.1ml for three total immunizations, spaced one week apart. Mice injected subcutaneously with either DC or PBS without epitope peptide were incubated as negative control mice. 7 days after the last immunization, spleens (sensitized DC group, empty DC group, PBS group) of each group of mice were picked up by aseptic operation, and erythrocytes were lysed to prepare single cell suspensions. Spleen cell suspensions (1X 10 ⁶/ml) were added to ELISPOT pre-coated plates (MabTech Inc), 200. Mu.l per well, and stimulated with the corresponding epitope peptide (final concentration 20. Mu.g/ml) for 24h. Spleen cells stimulated with PMA (daryou 2030421) were used as positive stimulation control wells. After the culture is finished and the cells are emptied, the cells are washed by PBS for 5 times, then the color development liquid is added for color development, and after the cells are sufficiently dried, the counting and the statistical analysis are performed by a plate reader.

Results: the Elispot assay results are shown in FIG. 2. The results indicate that SMp-11 (epitope peptide 5) sensitized DC immunized mice can secrete significantly increased amounts of IFN-gamma spots under stimulation of SMp-11 epitope peptide compared to negative control (non-sensitized DC or PBS immunized mice). While the other four epitope peptides (epitope peptides 1,3, 7, 8) showed no significant differences in secretion compared to the control.

Also, while epitope peptide 8 comprises 7 identical contiguous amino acids with epitope peptide 5, and has high similarity in sequence, its effect on inducing a specific immune response is quite opposite: the results showed that epitope peptide 8 failed to induce a specific immune response and was not a specific epitope; the epitope peptide 5 can not only efficiently induce immune response, but also has excellent specificity.

Conclusion: in view of the complexity of in vivo functions of organisms, whether the polypeptide with high affinity to HLA-A x 0201 molecules can sensitize DCs or not and the ability of the sensitized DCs to induce immune responses in vivo are unpredictable, and the specific immune induction effect of the sensitized DCs needs to be verified by in vivo tests. The in vivo test of the embodiment proves that the SMp-11 epitope polypeptide can effectively sensitize DC, and the sensitized DC can extremely remarkably induce specific IFN-gamma immune response aiming at SMp-11 epitope in mice.

Example 3: characterization of SMp-11 epitope peptide-sensitized DC cells

Peripheral blood mononuclear cells of healthy humans were isolated, PBMC were resuspended in RPMI1640 serum-free medium, sampled and counted, cell densities were adjusted to 5X 10 ⁶/ml, 2ml were plated per well in 6-well plates and incubated overnight at 37℃at 5% CO ₂ concentration. Shaking and blowing off cells (mainly lymphocytes) which are not adhered to the wall in the next day, and collecting and freezing; the adherent cells were monocytes, and 2ml of complete medium containing human recombinant GM-CSF (50 ng/ml) and human recombinant IL-4 (10 ng/ml) was added to each well of a six well plate, and 2ml of the same medium was fed every other day. Immature dendritic cells (DENDRITIC CELLS, DC) induced to differentiate by monocytes are collected until the fifth day, SMp-11 epitope peptide is added to 20 mug/ml, human TNF-alpha is added to 10ng/ml for stimulating maturation at the sixth day, and SMp-11 epitope peptide sensitized mature DC cells are obtained after culturing until the eighth day.

Mature DC cells were resuspended to 1X 10 ⁶/100. Mu.l in PBS, 1. Mu.l each of FITC-CD80, PE-CD83, APC-CD86 flow-through antibodies (Biolegend Inc) were added, mixed well, incubated at 4℃for 30 minutes in the absence of light, washed once with PBS and examined by flow cytometry for expression of CD80, CD83, CD86 molecules characteristic of mature DC (FIG. 3).

Results: the expression of the surface molecules characteristic of the SMp-11 epitope peptide sensitized DC cells is shown in figure 3, and the result shows that the surface CD80, CD83 and CD86 characteristic molecules of the SMp-11 epitope peptide sensitized DC cells after stimulation and maturation are all expressed highly.

Conclusion: mature SMp-11 peptide sensitized DC cells were successfully induced in vitro.

Example 4: SMp-11 specific humanized CTL induction culture and specific killing effect detection

Autologous T cells and dendritic cells were isolated from commercially available human PBMC cells (s 2001002) using conventional methods, and the cultured T cells were stimulated once a week with mature SMp-11 sensitized autologous dendritic cells, and co-stimulated three times. After three weeks of culture and expansion, effector cells were collected and tested for their specific killing effect by CFSE/7-AAD.

T2 cells loaded with SMp-11, T2 cells loaded with OVA polypeptide (SEQ ID NO:13, SIINFEKL, i.e.OVA _257-264) and empty T2 cells not loaded with polypeptide were used as target cells, respectively, labeled with CFSE working solution for 15 minutes (200. Mu.l CFSE/2X 10 ⁶ cells) at 37℃were incubated, washed once with complete medium, and the cell concentration was adjusted to 1X 10 ⁵ cells/ml with complete medium, and added to 96-well round bottom plates at 100. Mu.l per well.

Effector cells were added at three different target ratios of 10:1, 5:1, 2.5:1, loaded SMp-11 without effector cells, OVA _257-264 or empty T2 cells were used as background control, incubated for 4 hours at 37 ℃, collected by centrifugation, labeled with 7-AAD working solution for 15 minutes at 4 ℃, resuspended after washing twice with PBS, and CFSE and 7-AAD fluorescent signals were detected by flow cytometry.

The calculation formula of the killing rate is as follows:

killing rate (%) = experimental group CFSE-7AAD double positive cells (%) -background group CFSE-7AAD double positive cells (%)

Results: the result of the CFSE/7-AAD killing detection method is shown in figure 4, and the result shows that SMp-11 sensitized DC can effectively induce T lymphocytes, so that the T lymphocytes have extremely obviously improved killing efficiency on target cells loaded with SMp-11 epitope peptide compared with a control group (simple T2 cells or T2 cells loaded with irrelevant peptide).

Conclusion: SMp-11 sensitized DC induced effector T lymphocytes with excellent SMp-11 epitope specific killing.

Example 5: detection of IFN-gamma cytokine secretion by SMp-11 sensitized DC-induced specific human CTL

Effector T cells induced in vitro as in the previous method were resuspended at 2X 10 ⁶ per ml with complete medium and 100 μl per well was added to the ELISPOT pre-coated plate. The cells were adjusted to a cell concentration of 1X 10 ⁶ cells/ml with complete medium using SMp-11-loaded T2 cells, OVA polypeptide-loaded (SIINFEKL, OVA _257-264) -loaded T2 cells and polypeptide-unloaded empty T2 cells as stimulation cells, respectively, and ELISPOT pre-coated plates (MabTech Inc) were added at different effective target ratios (1:0.1, 1:0.25, 1:0.5). And taking the loaded SMp-11, OVA _257-264 or empty T2 cells without effector cells as a background control group, incubating for 24 hours at 37 ℃, cleaning the cells for 5 times by using PBS after evacuating the cells, adding a chromogenic solution for developing color, and counting the number of the IFN-gamma spots displayed by using a plate reader and carrying out statistical analysis after full drying. Based on the proportion of CD8 positive T cells and the number of IFN-gamma spots in the effector T cells, the proportion of CTL cells secreting IFN-gamma was calculated.

The ratio of IFN-gamma secreting CTL cells was calculated as follows:

results: the Elispot assay results are shown in FIG. 5. The results show that induced SMp-11-specific CTLs have significantly improved IFN- γ secretion under stimulation of target cells loaded with SMp-11 epitope peptide over control groups (T2 cells alone or T2 cells loaded with unrelated peptide), and that the proportion of ifnγ -secreting CTLs is up to 0.87% under stimulation of the lowest proportion of stimulated cells.

Conclusion: SMp-11 sensitized DC induced SMp-11 specific CTL can specifically and efficiently secrete IFN-gamma under the stimulation of target cells.

While the preferred embodiments of the present application have been described in detail, the present application is not limited to the embodiments, and various equivalent modifications and substitutions can be made by those skilled in the art without departing from the spirit of the present application, and these equivalent modifications and substitutions are intended to be included in the scope of the present application as defined in the appended claims.

Sequence information

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Claims (10)

1. Dendritic cells targeted to the novel coronavirus (SARS-CoV-2) M protein.

2. The dendritic cell of claim 1, wherein the dendritic cell is sensitized by an epitope polypeptide having a sequence as shown in FLWLLWPVT (SEQ ID NO: 5).

3. The dendritic cell of claim 2, wherein the epitope polypeptide is an HLA-A2 restricted epitope peptide; and/or

The epitope polypeptide has an affinity coefficient for the cell surface HLA-A 0201 molecule of at least 2.0, e.g. at least 2.2, at least 2.5.

4. The dendritic cell of claim 1, wherein the dendritic cell is a mature dendritic cell, e.g., expressing a surface molecule characteristic of a mature dendritic cell (e.g., CD80, CD83, CD 86); and/or

The dendritic cell surface presents SARS-CoV-2M protein antigen; and/or

The dendritic cells have enhanced antigen presenting capacity compared to a non-sensitized reference dendritic cell; and/or

The dendritic cells enhance secretion of cytokines (e.g., interferons such as IFN-gamma) or chemokines; and/or

The dendritic cells are effective in inducing effector cells targeting the novel coronavirus (SARS-CoV-2) M protein.

5. The dendritic cell of claim 1, wherein the dendritic cell is derived from bone marrow cells, umbilical cord blood cells, peripheral blood mononuclear cells; and/or

The dendritic cells are derived from mammals, e.g., humans, non-human primates, mice.

6. An article of manufacture comprising the dendritic cell of any one of claims 1-5.

7. The article of claim 6, wherein the article is a cellular article; and/or

The form of the dendritic cells is selected from the group consisting of: dendritic cells alone or in combination with viral therapeutic agents, cytotoxic agents, radionuclides, toxins, detectable labels, prodrug activating enzymes.

8. Use of a dendritic cell according to any of claims 1 to 5, a preparation according to any of claims 6 to 7 for the preparation of a product for the detection, diagnosis, prevention and/or treatment of SARS-CoV-2 related diseases.

9. The use of claim 8, wherein the product is selected from the group consisting of: dendritic cell vaccines, adoptive cell therapy drugs, effector cell stimulators, or combinations thereof, e.g., the product comprises the dendritic cells or specific T cells induced with the dendritic cells.

10. A method of preparing the dendritic cell of any one of claims 1-5, the method comprising:

(a) Providing a SARS-CoV-2M protein epitope polypeptide as set forth in any one of claims 1-3;

(b) Sensitizing dendritic cells or precursors thereof with the epitope polypeptides.