Novel coronavirus hla-a2 restricted epitope peptide and application thereof

Abstract

Provided herein are SARS-CoV-2 HLA-A2 restricted epitope peptides and uses thereof. Specifically, provided herein is an isolated polypeptide having an amino acid sequence of FLWLLWPVT (SEQ ID NO: 5), which is a SARS-CoV-2 HLA-A2 restricted epitope polypeptide. Also provided herein are complexes comprising the polypeptide, articles of manufacture, and uses thereof. The active substances and products related to the present application can be used for the detection, diagnosis, prevention, and / or treatment of SARS-CoV-2 related diseases.

Description

Technical Field

[0001] This invention relates to the fields of immunology and biomedicine. More specifically, this application relates to SARS-CoV-2 virus HLA-A2 restricted epitope peptides (e.g., the peptide of SEQ ID NO:5), as well as said epitope peptides and their complexes, products and their applications, such as in the preparation of products for the detection, diagnosis, prevention and / or treatment of SARS-CoV-2 related diseases. Background Technology

[0002] The main clinical symptoms of COVID-19 patients infected with SARS-CoV-2 include fever, cough, and shortness of breath. Laboratory tests often reveal multiple ground-glass opacities in the lungs. The condition of some patients can deteriorate rapidly, leading to serious complications, including acute respiratory distress syndrome, acute kidney injury, secondary infections, and cytokine storms. Some patients ultimately die from respiratory failure, multiple organ dysfunction, or shock.

[0003] The pathogen causing the disease, SARS-CoV-2, has been identified in the laboratory as a newly discovered beta coronavirus. It belongs to the same genus as SARS-CoV, the pathogen of the 2003 SARS outbreak, and shares 79% genetic similarity. Its diameter is 60–140 nm, and it possesses an envelope and a single-stranded, positive-sense RNA genome. The SARS-CoV-2 genome has 5' and 3' untranslated regions flanking it. The 5' end includes two relatively long open reading frames (ORFs) encoding 16 non-structural proteins; the remaining portion of the genome near the 3' end mainly encodes structural proteins and other accessory proteins. The virus's structural proteins primarily include the spike protein (S protein), membrane glycoprotein (M protein), envelope glycoprotein (E protein), and nucleocapsid protein (N protein).

[0004] The S protein, which binds to host cells and mediates viral infection, is currently the primary antigenic protein referenced in antibody and vaccine development. However, with the prolonged pandemic, SARS-CoV-2 has accumulated mutations in the host, especially in its structural proteins, leading to the emergence of several adaptively superior SARS-CoV-2 mutant strains, such as Omeprón. These mutant strains typically possess stronger infectivity or pathogenicity, and the mutations may alter antigenic properties, thereby affecting the effectiveness of preventative vaccines and therapeutic antibodies against these mutant strains and causing viral immune evasion within the body. Gene sequence analysis shows that structural protein mutations are mainly concentrated in the S and N proteins, posing a significant challenge to vaccines or antibodies developed based on S protein antigenic information.

[0005] The M protein is essentially a transmembrane protein characterized by three domains: an N-terminal extracellular domain, a triple-transmembrane domain, and an internal C-terminal domain. It plays a crucial role in viral morphogenesis and maintenance and is the most abundant glycoprotein in viral particles. Crucially, the M gene is relatively conserved, and the mutation rate of the M protein is far lower than that of the S protein. Vaccines or antibodies developed based on the M protein are less likely to experience reduced efficacy due to viral mutations. Therefore, targeting the M protein for immunization will, on the one hand, induce a specific immune response against the viral M protein, enabling the body to effectively clear the virus and providing a means for the prevention and treatment of SARS-CoV-2. On the other hand, its relatively conserved low mutation rate will further prevent viral immune escape due to mutations, resulting in a broader-spectrum effect.

[0006] Synthetic peptide vaccines are a new type of vaccine that has emerged in recent years with advancements in molecular biology and immunology. They can induce a specific immune response in the body, and have mild side effects and good safety profiles, making them a hot research area in vaccine development. They are widely used in anti-tumor and antiviral immunotherapy. Several epitope-based peptide vaccines are currently in clinical trials or have been marketed.

[0007] HLA-A*0201 is a class I MHC molecule with a high distribution in the Chinese population, exhibiting a positive rate between 40% and 60%, ranking first among all subgroups of class I MHC molecules and making it a preferred molecule for vaccine design. Currently, several HLA-A*0201-restricted CTL epitopes have been identified, some of which have shown good clinical efficacy. However, due to the diversity and complexity of cellular epitopes, further in-depth research targeting specific proteins is needed to develop epitope peptides for immunization or therapeutic applications.

[0008] T2 cells are one of the tool cells used to determine the binding affinity between epitopes and HLA-A*0201 molecules. They are HLA-A*0201 cell lines that are deficient in antigen-presenting transporters. The surface of these cells expresses only HLA-A*0201 molecules that do not contain endogenous antigen molecules, so the degree of binding between them and the target peptide can be used to determine the affinity between HLA-A*0201 molecules and the target epitope.

[0009] There remains an urgent need in this field to develop pathways, products, and methods that can effectively prevent and treat SARS-CoV-2 and related diseases. Summary of the Invention

[0010] This application provides an HLA-A2-restricted epitope polypeptide molecule targeting the M protein, one of the main structural proteins of the SARS-CoV-2 virus. This molecule can bind to HLA-A2 with high affinity and has M protein specificity, and can be effectively applied to the preparation and development of downstream products.

[0011] In some aspects of this document, an isolated polypeptide is provided, comprising the amino acid sequence FLWLLWPVT (SEQ ID NO:5). In some embodiments, the amino acid sequence of the isolated polypeptide of this application is FLWLLWPVT (SEQ ID NO:5).

[0012] In some embodiments, the isolated polypeptide is a novel coronavirus SARS-CoV-2 epitope peptide, and the polypeptide is an HLA-A2 restricted epitope peptide.

[0013] In some embodiments, the isolated polypeptide is an M protein epitope peptide of SARS-CoV-2, and the polypeptide is an HLA-A*0201 restricted epitope peptide.

[0014] In some embodiments, the affinity coefficient between the isolated peptide and the HLA-A*0201 molecule on the cell surface is at least 2.0, for example, at least 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, or 4.0.

[0015] In some aspects thereof, a complex comprising the epitope polypeptide described in this application is provided.

[0016] In some embodiments, the complex is selected from: proteins or fusion proteins or recombinant proteins containing the epitope polypeptide of this application, and antigen-presenting cells loaded with the epitope polypeptide of this application.

[0017] In some embodiments, the antigen-presenting cells are selected from one or more of the group consisting of dendritic cells, macrophages, B cells, fibroblasts, endothelial cells, or cells obtained by artificial modification of any of the foregoing.

[0018] In some aspects of this document, an isolated nucleic acid molecule is provided that encodes the epitope polypeptide of this application or a protein, fusion protein, or recombinant protein containing the epitope polypeptide of this application.

[0019] In some aspects of this document, an article comprising the epitope peptide and / or complex described herein is provided.

[0020] In some embodiments, the article of manufacture may further comprise an acceptable carrier or excipient.

[0021] In some embodiments, the article is a composition or a kit.

[0022] In some aspects thereof, uses of the peptides, complexes and / or articles of this application for the preparation of one or more products selected from the group consisting of: dendritic cells sensitized with the peptide, specific immune effector cells that are specific to the peptide, targeted drugs that target the peptide, vaccines that target the peptide, and detection reagents or kits that target the peptide.

[0023] In some implementations, the product is used for the detection, diagnosis, prevention, and / or treatment of SARS-CoV-2 related diseases.

[0024] In some aspects of this document, a method is provided for preparing a product selected from the group consisting of: peptide-sensitized dendritic cells, specific immune effector cells that are specific to peptides, targeted drugs targeting peptides, vaccines targeting peptides, and detection reagents or kits targeting peptides, the method comprising:

[0025] (a) Provide the polypeptides, complexes and / or articles described herein;

[0026] (b) Using the polypeptide, complex or article described in (a) for sensitizing the dendritic cells, stimulating the immune effector cells, or preparing the drug, vaccine, diagnostic reagent or kit.

[0027] In some implementations, the resulting product is used for the detection, diagnosis, prevention, and / or treatment of SARS-CoV-2-related diseases.

[0028] In some aspects of this article, sensitized dendritic cells, specific immune effector cells, targeted drugs, targeted vaccines, targeted detection reagents or kits prepared using the methods described herein are provided.

[0029] In some embodiments, the sensitized dendritic cells, specific immune effector cells, targeted drugs, targeted vaccines, targeted detection reagents or kits are used for the detection, diagnosis, prevention and / or treatment of SARS-CoV-2 related diseases.

[0030] Those skilled in the art can combine the foregoing technical solutions and features in any way without departing from the inventive concept and protection scope of this invention. Other aspects of this invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0031] The present invention will be further described below with reference to the accompanying drawings, which are shown only for illustrating embodiments of the present invention and are not intended to limit the scope of the present invention.

[0032] Figure 1The SMp-11 epitope peptide exhibits a high affinity for the HLA-A*0201 molecule. Table 1 shows the flow cytometry results predicting the binding affinity between the epitope peptide and the HLA-A*0201 molecule. A higher fluorescence coefficient indicates a higher affinity between the epitope peptide and HLA-A*0201 (generally, a fluorescence coefficient greater than 1 indicates that the epitope peptide has a high affinity for HLA-A*0201).

[0033] Figure 2 Epitope-specific immune responses induced in transgenic mice immunized with murine DCs sensitized with epitope peptides. This figure represents the Elispot results analysis (“****”, P<0.0001, “ns” = no significant difference).

[0034] Figure 3 Human dendritic cells (DCs) sensitized with the SMp-11 epitope peptide were successfully induced and cultured in vitro. The figure shows the expression intensity of maturation marker molecules on the surface of DC cells after SMp-11 induction by flow cytometry.

[0035] Figure 4 Human DCs sensitized with SMp-11 induced effector T lymphocytes exhibited SMp-11 epitope-specific killing activity. This figure shows the CFSE / 7-AAD flow cytometry analysis of the killing effect of SMp-11-specific CTLs ("***", P<0.001; "****", P<0.0001, "ns" = no significant difference).

[0036] Figure 5 SMp-11-specific human CTLs showed a high proportion in sensitized dendritic cell (DC)-induced effector T cells. This figure shows the results of flow cytometry analysis using the Tetramer method (Tetramer loaded with OVA served as the flow cytometry control).

[0037] Figure 6 SMp-11-specific human CTLs can specifically secrete the cytotoxic cytokine IFN-γ upon stimulation of target cells. This figure shows the Elispot results analysis ("****", P<0.0001, "ns" = no significant difference) (target cells loaded with irrelevant peptide OVA served as the control group). Detailed Implementation

[0038] This application describes the discovery and development of a high-affinity binding agent for the M protein, one of the main structural proteins of the SARS-CoV-2 virus, which can be effectively applied to the preparation and development of downstream products.

[0039] Based on in-depth analysis of the SARS-CoV-2 novel coronavirus M protein, we selectively synthesized several SARS-CoV-2 candidate antigenic peptides that may bind to HLA-A*0201 and induce the production of CTLs in the body. Through T2 peptide binding assays, we screened epitope peptides with strong affinity for HLA-A*0201 and evaluated their immunogenicity. We found that these peptides can induce specific, HLA-A*0201-restricted cytotoxic T lymphocytes in the peripheral blood of healthy individuals with HLA-A*0201, and are immunogenic peptides that are naturally processed and presented by cells. The identification of HLA-A2-restricted cytotoxic T lymphocyte epitope peptides derived from the SARS-CoV-2 novel coronavirus M protein is of great significance not only for the study of the pathogenesis of SARS-CoV-2 but also for the development of vaccines and therapeutic agents.

[0040] Features mentioned in this invention or in the embodiments may be combined. All features disclosed in this specification may be used in any compositional form, and each feature disclosed in the specification may be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0041] All numerical ranges provided in this document are intended to clearly include all numerical values ​​falling between the endpoints of the range and the range of numerical values ​​between them. For example, 1 to 3 includes endpoints 1 and 3, specific integer numerical points 2 and non-integer numerical points (e.g., but not limited to: 1.2, 1.5, 1.8, 2.1, 2.3, 2.4, 2.8, etc.), and its subranges (e.g., but not limited to: 1 to 2, 2 to 3, 1 to 1.2, 1.5 to 1.8, etc.).

[0042] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0043] As used herein, “invention polypeptide”, “specific (poly)peptide”, “HLA-A2 restricted epitope (poly)peptide” and “SARS-CoV-2HLA-A2 restricted epitope (poly)peptide” are used interchangeably and refer to epitope peptides derived from the M protein of SARS-CoV-2 that have a high affinity for HLA-A2.

[0044] The functional epitope peptides of this application may include or be the peptide of SEQ ID NO:5. The peptides of this invention may be naturally purified products, chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher animals, insects, and mammalian cells) using recombinant technology.

[0045] As used herein, the terms "epitope polypeptide coding sequence" or "epitope polypeptide coding molecule" refer to the sequence encoding the epitope polypeptide or its active fragment as described herein. After obtaining the amino acid sequence of the epitope polypeptide of this invention, those skilled in the art can obtain its coding sequence using methods known in the art, and can optimize the sequence. For example, the epitope polypeptide coding sequence can be obtained from the full-length coding sequence or CDS sequence corresponding to the SARS-CoV-2M protein. The coding sequences of this application may also include molecules that hybridize to these sequences under stringent conditions, or family gene molecules that are highly homologous to the aforementioned molecules.

[0046] As used herein, the term "strict conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2 × SSC, 0.1% SDS, 60°C; or (2) hybridization with the addition of a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the similarity between the two sequences is at least 50%, preferably 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, more preferably 95%. For example, the sequence may be a complementary sequence to the sequence defined in (a).

[0047] The polypeptides of the present invention can also exist in various forms as needed. The epitope polypeptides of the present invention can exist alone, or can be coupled or fused with other proteins or polypeptides to form polypeptide complexes, or can be loaded into antigen-presenting cells, or exist in the above forms in compositions or kits and other products.

[0048] In some embodiments, the epitope polypeptides described herein may be included in a fusion peptide, such as a portion fused thereto including: a viral or host-derived protein, transferrin (Fn), HIV p24, the stem of an enveloped virus, such as influenza HA2, HIV gp41, an antibody Fc fragment, GM-CSF, IL-21, CD40L, or CD40 antibody.

[0049] In some embodiments, the fusion peptide may further include elements such as a signal peptide, a linker peptide, and a tag. For example, the signal peptide may be selected from: the protein itself, CD33, CD8, CD16, mouse IgG1 antibody, or influenza HA. ​​The linker peptide may be selected from: (G4S)3, (G4S... n,GSAGSAAGSGEF,(Gly)6,EFPKPSTPPGSSGGAP,KESGSVSSEQLAQFRSLD,(Gly)8,EGKSSGSGSESKST. Tags can be selected from: His-tag, AviTag, Calmodulin tag, polyglutamate tag, E-tag, FLAG tag, HA-tag, Myc-tag, S-tag, SBP-tag, Sof-tag 1, Sof-tag3, Strep-tag, TCtag, V5 tag, T7 tag, VSV tag, Xpress tag, 3X FLAG tag, Isopep tag, Spytag, Snoop tag and PNE tag.

[0050] In some embodiments, the epitope peptide of the present invention can be coupled to a protein of molecular weight such as BSA to form a peptide conjugate. Typically, the conjugate consists of a peptide, a cross-linking agent, and BSA, wherein the cross-linking agent is preferably glutaraldehyde or EDAC. For example, the epitope peptide of the present invention can be loaded onto antigen-presenting cells. In some embodiments, the antigen-presenting cells are one or more selected from the group consisting of dendritic cells, macrophages, B cells, fibroblasts, endothelial cells, or cells obtained by artificial modification of any of the foregoing.

[0051] The peptides, complexes, and / or articles described in this application can be used to prepare a variety of products. These products may include, but are not limited to, one or more selected from the group consisting of: dendritic cells sensitized with the peptide, specific immune effector cells that are specific to the peptide, targeted drugs that target the peptide, vaccines that target the peptide, detection reagents or kits that target the peptide, for example, using the peptide directly as an immunogen to prepare immune compositions or pharmaceutical compositions.

[0052] The present invention also provides a medicament, pharmaceutical composition or kit containing an effective amount of the epitope polypeptide, complex or article of the present invention, or a product made from the epitope polypeptide, complex or article, and a pharmaceutically or immunologically acceptable carrier.

[0053] In a preferred embodiment, the pharmaceutical composition can be used to detect, diagnose, prevent, and / or treat diseases associated with SARS-CoV-2, chronic diseases caused by SARS-CoV-2, and / or their symptoms. For example, the pharmaceutical compositions of the present invention can be used to prevent or treat infectious diseases or symptoms caused by the novel coronavirus, such as lung or other tissue damage, complications, multiple organ failure, etc.

[0054] In some embodiments, the products described herein can be used to prevent, eliminate, or alleviate novel coronavirus infection or at least one of its symptoms in a subject, such as respiratory symptoms (e.g., nasal congestion, sore throat, hoarseness), headache, cough, sputum, fever, rales, wheezing, difficulty breathing, pneumonia caused by infection, severe acute respiratory syndrome, kidney failure, etc.

[0055] As used herein, the terms “containing” or “including” include “comprising,” “substantially consisting of,” and “consisting of.” As used herein, the term “acceptable” refers to a substance that is suitable for use in humans and / or animals and / or other subjects (such as cells) without excessive adverse effects (such as toxicity, irritation, and allergic reactions), i.e., 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 (such as cells) and is acceptable to the subject.

[0056] As used herein, the term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of therapeutic agents, which may include a variety of excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity upon administration. Suitable carriers are well known to those skilled in the art, and a thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0057] Acceptable carriers in the composition may contain liquids such as water, saline, glycerol, and ethanol. Additionally, these carriers may contain auxiliary substances such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting agents or emulsifiers, flavoring agents, pH buffers, etc. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, with a pH usually around 5-8, preferably around 6-8.

[0058] The active substance in the composition of the present invention accounts for 0.001 to 99.9 wt% of the total weight of the composition; preferably 1 to 95 wt% of the total weight of the composition, more preferably 5 to 90 wt%, and more preferably 10 to 80 wt%. The balance is a pharmaceutically acceptable carrier and other additives.

[0059] As used herein, the term "unit dosage form" refers to a dosage form in which the compositions of the present invention are prepared for a single dose for ease of administration, including but not limited to various solid dosage forms (such as tablets), liquid dosage forms, capsules, and sustained-release formulations.

[0060] In another preferred embodiment of the invention, the composition is in a single dosage form or multiple dosage forms. In another preferred embodiment of the invention, 1 to 6 doses of the composition of the invention are administered daily, preferably 1 to 3 doses; most preferably, the daily dose is 1 dose.

[0061] It should be understood that the effective dose of the active substance used can vary depending on the severity of the condition of the patient being treated. The specific dosage is determined based on the individual patient's circumstances (e.g., weight, age, physical condition, and desired outcome), within the judgment of a skilled physician.

[0062] The compositions of the present invention can be in solid form (e.g., granules, tablets, lyophilized powder, suppositories, capsules, sublingual tablets) or liquid form (e.g., oral liquid) or other suitable forms. Administration routes may include: (1) direct naked protein injection; (2) linking the active substance to a transferrin / poly-L-lysine complex to enhance its biological effects; (3) forming a complex of the active substance with positively charged lipids to overcome the difficulty of crossing the cell membrane caused by the negative charge of the phosphate backbone; (4) liposome encapsulation; (5) binding to cholesterol to increase its cytoplasmic retention time by 10 times; (6) specific transport to target tissues and cells using immunoliposomes; (7) in vitro transfection; and (8) electroporation to introduce the active substance into target cells.

[0063] Example

[0064] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make appropriate modifications and variations to the present invention, and such modifications and variations are all within the scope of the present invention.

[0065] Experimental methods not specifically described in the following examples can be performed using conventional methods in the art, such as those described in *Molecular Cloning: A Laboratory Manual* (3rd edition, New York: Cold Spring Harbor Laboratory Press, 1989) or according to the conditions recommended by the supplier. DNA sequencing methods are conventional in the art and can also be provided by commercial companies.

[0066] Unless otherwise stated, percentages and parts are by weight. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0067] Example 1: Affinity determination of epitope peptides with HLA-A*0201 molecules

[0068] Peptide binding assays were used to screen for epitope peptides with high affinity for HLA-A*0201. First, T2 cells (Fuheng Biotechnology, FH0150) were collected, washed three times with serum-free 1640 medium, and the cell concentration was adjusted to 2 × 10⁻⁶ cells / year. 6 Cells were seeded at 1 ml / well in 24-well plates. They were then incubated with 50 μM of the candidate peptide and 3 μg / ml of β2 microglobulin at 37°C in a 5% CO2 incubator for 18 h. After incubation, the cells were washed three times with ice-cold PBS, and then incubated with PE-labeled HLA-A2-specific flow cytometry antibody (Biolegend Inc.) at 4°C for 30 min. After washing with PBS, the mean fluorescence intensity was measured by flow cytometry. The HLA-A2-restricted influenza virus epitope peptide GILGFVFTL was used as a positive control, and unstimulated T2 cells were used as a background control.

[0069] Results Interpretation: Flow cytometry was used to detect the binding of peptides to HLA-A*0201 molecules. This is based on the fact that the binding of exogenous peptides to MHC class I molecules on the surface of T2 cells increases the expression level of MHC class I molecules on the cell surface. The stronger the binding, the more MHC class I molecules can be detected. The average fluorescence intensity was used as the detection index. The results were measured by the fluorescence coefficient (FI). A peptide with FI > 1 was considered a high-affinity epitope.

[0070] The formula for calculating the fluorescence coefficient (FI) is as follows:

[0071]

[0072] Following this method, high-affinity HLA-A2 epitope peptides were screened from the membrane glycoprotein M protein (SEQ ID NO:10), spike glycoprotein S protein (SEQ ID NO:11), and envelope protein E protein (SEQ ID NO:12) of SARS-CoV-2 coronavirus. The average fluorescence intensity and fluorescence coefficient of the exemplary epitope peptides used in the screening are shown in Table 1 and [Table data missing]. Figure 1 :

[0073] Table 1. Fluorescence coefficients (FI) of different epitope peptides after binding to HLA-A*0201 molecules on the surface of T2 cells by flow cytometry.

[0074]

[0075] Results: The affinity results of exemplary candidate epitope peptides with HLA-A*0201 molecules are shown in Table 1. The results show that the 1st, 3rd, 5th, 7th and 8th peptide epitopes (named epitope peptide 1, epitope peptide 3, epitope peptide 5 (also known as SMp-11), epitope peptide 7 and epitope peptide 8, respectively) ranked first, with average fluorescence intensity greater than 10000 and fluorescence coefficients (FI) greater than 1, even reaching 2.0 or higher; among them, the FI of epitope peptide 5 (also known as SMp-11) reached as high as 2.73.

[0076] Conclusion: Through screening, peptide epitopes with high affinity for the HLA-A*0201 molecule were obtained, and the top five peptide epitopes (1, 3, 5, 7, 8) were selected for further screening and identification.

[0077] Example 2: Detection of IFN-γ secretion in spleen cells of HLA-A2 transgenic mice sensitized with epitope peptides

[0078] Based on the five potential polypeptide epitopes (epitaxes 1, 3, 5, 7, and 8) screened in Example 1, their immunogenicity was further tested in HLA-A2 transgenic mice. HLA-A2.1 / K was prepared using conventional methods. b Dendritic cells (DCs) derived from bone marrow of transgenic mice (Jackson Laboratory, 003475). Mice were euthanized by cervical dislocation, and the femur and tibia were removed. PBS was drawn into the bone marrow cavity using a 1 ml syringe, and the needle was inserted to flush out the bone marrow. Bone marrow erythrocytes were then lysed with Tris-NH4Cl solution. After centrifugation and discarding the supernatant, DCs were induced and cultured in 1640 medium containing 10% FBS, 1 ng / ml mouse IL-4 (PeproTech, 214-14-5UG), and 10 ng / ml mouse GM-CSF (PeproTech, 315-03-50UG). DCs cultured to day 5 were collected and the cell concentration was adjusted to 1×10⁶ cells / day. 6 Add epitope peptides (final concentration 20 μg / ml) to each cell, and continue culturing in a 37℃, 5% CO2 incubator until day 6. Add TNF-α (30 ng / ml) to stimulate maturation, and continue culturing until day 8 to obtain epitope peptide-sensitized DC cells.

[0079] Collect peptide-sensitized DCs, wash three times with PBS, and adjust the cell concentration to 1×10⁻⁶. 7 0.1 ml / ml was injected subcutaneously into the abdomen of each male transgenic mouse, with three immunizations administered one week apart. Mice that were not incubated with DCs or PBS at the same time served as negative controls. Seven days after the last immunization, the spleens of each group of mice (sensitized DC group, empty DC group, PBS group) were aseptically harvested, and red blood cells were lysed to prepare single-cell suspensions. The spleen cell suspensions (1×10⁻⁶ cells / ml) were then used to prepare single-cell suspensions. 6Cells were added to an ELISPOT pre-coated plate (MabTech Inc.) at a concentration of 200 μl per well, and the corresponding epitope peptide (final concentration 20 μg / ml) was added to stimulate culture for 24 h. Spleen cells stimulated with PMA (Dayou, 2030421) were used as positive control wells. After culture, cells were drained, washed five times with PBS, and then chromogenic buffer was added for color development. After thorough drying, cells were counted and statistically analyzed using a plate reader.

[0080] Results: Elispot test results are as follows Figure 2 As shown in the results, compared with the negative control group (unsensitized DCs or PBS-immunized mice), SMp-11 (episode peptide 5)-sensitized DC-immunized mice showed a significantly increased number of IFN-γ spots secreted by their spleen cells upon stimulation with the SMp-11 epitope peptide. However, the secretion of the other four epitope peptides (episode peptides 1, 3, 7, and 8) showed no significant difference compared to the control.

[0081] Furthermore, although epitope peptide 8 and epitope peptide 5 contain seven identical consecutive amino acids and have high sequence similarity, their effects in inducing specific immune responses are completely opposite: the results show that epitope peptide 8 cannot induce a specific immune response and is not a specific epitope; while epitope peptide 5 can not only efficiently induce an immune response, but also this immune response has excellent specificity.

[0082] Conclusion: Given the complexity of biological processes, the ability of a polypeptide epitope with high affinity for HLA-A*0201 to effectively sensitize dendritic cells (DCs) and subsequently induce an effective immune response in vivo is unpredictable. In vivo testing is necessary to verify its DC sensitization and specific immune induction effects. The in vivo test in this embodiment verified that the SMp-11 epitope polypeptide of the present invention can effectively sensitize DCs and significantly induce a specific IFN-γ immune response against the SMp-11 epitope in mice.

[0083] Example 3: Characterization of DC cells sensitized with SMp-11 epitope peptide

[0084] Peripheral blood mononuclear cells (PBMCs) were isolated from healthy individuals, resuspended in RPMI 1640 serum-free medium, sampled, counted, and the cell density was adjusted to 5 × 10⁶ cells / mL. 6Cells were cultured at a density of 1 / ml, with 2 ml of the medium per well in a 6-well plate. The plates were incubated overnight at 37°C and 5% CO2. The next day, non-adherent cells (mainly lymphocytes) were shaken and detached, collected, and cryopreserved. Adherent cells, i.e., monocytes, were added to each well of the 6-well plate with 2 ml of complete culture medium containing recombinant human GM-CSF (50 ng / ml) and recombinant human IL-4 (10 ng / ml). The same medium was added every other day. On the fifth day, immature dendritic cells (DCs) induced from monocytes were collected, and SMp-11 epitope peptide was added to a concentration of 20 μg / ml. On the sixth day, human TNF-α was added to a concentration of 10 ng / ml to stimulate maturation. Mature DCs sensitized with SMp-11 epitope peptide were obtained on the eighth day of culture.

[0085] Take mature DC cells and resuspend them in PBS to a concentration of 1×10⁻⁶. 6 Add 1 μl each of FITC-CD80, PE-CD83, and APC-CD86 flow cytometry antibodies (Biolegend Inc.) to 100 μl of the sample, mix well, and incubate at 4°C in the dark for 30 minutes. After washing once with PBS, use flow cytometry to detect the expression of CD80, CD83, and CD86, characteristic surface molecules of mature dendritic cells (DCs). Figure 3 ).

[0086] Results: The expression of characteristic surface molecules in DC cells sensitized with SMp-11 epitope peptide was as follows: Figure 3 As shown, the results indicate that the characteristic molecules CD80, CD83, and CD86 on the surface of DCs sensitized by the SMp-11 epitope peptide after stimulation and maturation are highly expressed.

[0087] Conclusion: Mature SMp-11 peptide-sensitized DC cells can be successfully induced in vitro using the SMp-11 epitope peptide.

[0088] Example 4: Induction and culture of SMp-11-specific human CTLs and detection of their specific killing effect

[0089] Autologous T cells and dendritic cells were isolated from purchased human PBMCs (SaiLi Bio, s2001002) using standard methods. The cultured T cells were stimulated weekly with mature SMP-11-sensitized autologous dendritic cells for a total of three stimulations. After three weeks of culture and expansion, effector cells were collected, and their specific cytotoxic effect was detected using the CFSE / 7-AAD method.

[0090] T2 cells loaded with SMp-11 and OVA peptide (SEQ ID NO: 13, SIINFEKL, i.e. OVA) were used respectively. 257-264T2 cells and empty T2 cells without peptide loading were used as target cells and labeled by incubation with CFSE working solution at 37°C for 15 minutes (200 μl CFSE / 2 × 10⁻⁶). 6 (100 cells), wash once with complete culture medium, and adjust the cell concentration to 1×10⁻⁶ cells with complete culture medium. 5 Add 100 μl per well to a 96-well round-bottom plate.

[0091] Effector cells were added at three different effector-target ratios of 10:1, 5:1, and 2.5:1. The loading of SMP-11 and OVA without effector cells was different. 257-264 Alternatively, empty T2 cells were used as the baseline control group. The cells were incubated at 37°C for 4 hours, centrifuged to collect the cells, incubated with 7-AAD working solution at 4°C for 15 minutes, washed twice with PBS and resuspended. The fluorescence signals of CFSE and 7-AAD were detected by flow cytometry.

[0092] The formula for calculating the kill rate is as follows:

[0093] Kill rate (%) = CFSE-7AAD double-positive cells in experimental group (%) - CFSE-7AAD double-positive cells in baseline group (%)

[0094] Results: The results of the CFSE / 7-AAD kill detection method are as follows: Figure 4 As shown, the results indicate that SMp-11 sensitized DCs can effectively induce T lymphocytes, which exhibit a significantly higher killing efficiency against target cells loaded with SMp-11 epitope peptides compared to the control group (simple T2 cells or T2 cells loaded with unrelated peptides).

[0095] Conclusion: Epitope peptide SMp-11 can effectively sensitize dendritic cells (DCs) and induce the production of effector T lymphocytes with excellent SMp-11 epitope-specific killing activity.

[0096] Example 5: Detection of the proportion of SMp-11-specific human CTLs in in vitro induced effector T cells

[0097] Take 20 μl of peptide FLEX-T TM Tetramer monomer (Biolegend Inc; 280003) was mixed with 20 μl of SMp-11 epitope peptide (400 μM), and then irradiated with UV light (366 nm) on ice for 30 min. Following this, it was incubated at 37°C in the dark for 30 min. Then, 3.3 μl of PE-streptavidin (Biolegend Inc) was added, mixed, and incubated on ice in the dark for 30 min. Finally, 2.4 μl of blocking buffer was added and mixed (blocking buffer: 1.6 μl 50 mM D-biotin + 6 μl 10% (w / v) NaN3 + 192.4 μl PBS). The OVA-loaded peptide (SIINFEKL, OVA) was prepared using the same method.257-264 Tetramer (SEQ ID NO:13) was used as an irrelevant peptide control.

[0098] Effector T cells induced in vitro using the same method as in Example 4 were resuspended in PBS at a concentration of 2 × 10⁻⁶. 6 Add 2 μl of the prepared SMp-11-Tetramer or OVA-Tetramer to each 200 μl of the sample, mix well, and incubate at 4°C in the dark for 30 minutes. After washing with PBS, the proportion of SMp-11-specific CTLs bound to SMp-11-Tetramer is detected by flow cytometry.

[0099] Results: The Tetramer detection results for SMp-11-specific CTLs are as follows: Figure 5 As shown, the results indicate that in effector T cells induced by SMp-11 sensitized DCs, the proportion of SMp-11-specific CTLs reached 2.02%, while that in the unrelated peptide control group was only 0.37%.

[0100] Conclusion: Epitope peptide SMp-11 can effectively sensitize dendritic cells (DCs) and further induce a significant increase in the proportion of SMp-11-specific human CTLs in effector T cells, thereby inducing a specific T cell immune response against this SMp-11 epitope.

[0101] Example 6: Detection of IFN-γ cytokine secretion by SMp-11-specific human CTLs

[0102] Effector T cells induced in vitro using the same method as before were resuspended in complete culture medium at a concentration of 2 × 10⁻⁶. 6 100 μl of ELISPOT pre-coated plate was added per well to each well. The plate was then filled with T2 cells loaded with SMp-11 and cells loaded with OVA peptide (SIINFEKL, OVA). 257-264 T2 cells and empty T2 cells without peptide loading were used as stimulating cells, and the cell concentration was adjusted to 1×10⁻⁶ using complete culture medium. 6 Cells / ml were added to ELISPOT pre-coated plates (MabTech Inc.) at different effector-to-target ratios (1:0.1, 1:0.25, 1:0.5). No effector cells were added to the loaded SMp-11 or OVA. 257-264 Alternatively, empty T2 cells were used as a baseline control group. After incubation at 37°C for 24 hours, the cells were drained, washed five times with PBS, and then chromogenic solution was added for development. After thorough drying, the number of IFN-γ spots was counted using a plate reader and statistically analyzed. The proportion of CTL cells secreting IFN-γ was calculated based on the proportion of CD8-positive T cells and the number of IFN-γ spots in effector T cells.

[0103] The formula for calculating the proportion of CTL cells secreting IFN-γ is as follows:

[0104]

[0105] Results: Elispot test results are as follows Figure 6 As shown in the figure. The results indicate that induced SMp-11-specific CTLs exhibited significantly higher IFN-γ secretion under stimulation of target cells loaded with SMp-11 epitope peptides compared to the control group (T2 cells alone or T2 cells loaded with unrelated peptides), and the proportion of CTLs secreting IFN-γ was as high as 0.87% under stimulation with the lowest proportion of stimulated cells.

[0106] Conclusion: The epitope peptide SMp-11 can effectively sensitize dendritic cells (DCs) and further induce SMp-11-specific CTLs to specifically and efficiently secrete IFN-γ under target cell stimulation, thereby inducing a specific IFN-γ immune response against this SMp-11 epitope.

[0107] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

[0108] Appendix. Sequence Information

[0109]

[0110]

[0111]

Claims

1. A complex comprising a polypeptide with the amino acid sequence FLWLLWPVT (SEQ ID NO: 5), wherein the complex is an antigen-presenting cell loaded with the polypeptide, wherein the antigen-presenting cell is a dendritic cell.

2. An article comprising the complex as described in claim 1.

3. The article of claim 2, wherein, The product in question is a reagent kit.

4. Use of the complex of claim 1 or the article of any one of claims 2-3 in the preparation of a product, wherein the product is a specific immune effector cell with specificity for the polypeptide having the amino acid sequence FLWLLWPVT (SEQ ID NO: 5), wherein, The product is used for the detection, diagnosis, prevention and / or treatment of SARS-CoV-2.

5. A method for preparing a product, said product being specific immune effector cells that are specific to a polypeptide with the amino acid sequence FLWLLWPVT (SEQ ID NO: 5), said method comprising: (a) Providing the compound as claimed in claim 1 or the article as claimed in any one of claims 2 to 3; (b) Using the complex or product described in (a) to stimulate the immune effector cells, wherein the product is used for the detection, diagnosis, prevention and / or treatment of SARS-CoV-2.

6. Specific immune effector cells prepared by the method of claim 5, which are used for the detection, diagnosis, prevention and / or treatment of SARS-CoV-2.

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