Human respiratory coronavirus cross-reactive t cell epitope map and methods of delivery thereof

Abstract

The application discloses a human respiratory tract coronavirus cross-reactive T cell epitope map and a delivery method thereof. The application draws a cross-reactive T cell epitope map among 7 HCoVs, and further screens an epitope fragment with high conservation from the identified cross-epitopes, so as to provide a core theoretical basis and experimental basis for development of a broad-spectrum HCoVs immune target; then, a Venezuelan equine encephalitis virus replicon vaccine precision delivery system capable of expressing a single conservative epitope is constructed for the screened conservative epitope fragment, so that the targeted delivery of the single conservative cross-reactive T cell epitope is realized, and a key technical bottleneck of low cross-epitope delivery efficiency in the existing HCoVs vaccine is solved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a cross-reactive T-cell epitope map of human respiratory coronaviruses and its delivery method. Background Technology

[0002] The seven known human respiratory coronaviruses (HCoVs) continue to pose a serious threat to global public health and human health, and humans may still face the potential risk of infection from emerging coronaviruses in the future. HCoVs encode four structural proteins (spike protein (S), membrane protein (M), envelope protein (E), and nucleocapsid protein (N)) and varying numbers of accessory proteins. Multiple studies have shown that structural proteins can induce strong adaptive immune responses: on the one hand, they can induce high levels of humoral immune responses; on the other hand, they can induce long-term cellular immune response memory. For example, even 17 years after recovery from SARS-CoV infection, multispecific, long-acting T-cell immunity against structural protein N can still be detected in patients, laying an important immune foundation for providing long-term immune protection. Since the outbreak of the novel coronavirus (COVID-19), whether there is cross-reactive T-cell response among HCoVs has become a research hotspot. However, several scientific bottlenecks remain to be overcome in this field: First, no systematic analysis of the epitope maps of cross-reactive T cells among the seven HCoVs has been conducted, especially a systematic study based on clinical samples. Second, are there broad-spectrum cross-reactive T cell epitopes among different HCoVs that could support the design of broad-spectrum coronavirus vaccines? Existing vaccines often target the HCoV-S protein, failing to meet the needs of controlling infection with different HCoVs and emerging variants, making it difficult to construct a "one vaccine against multiple viruses" protection system. Third, there is currently a lack of precise delivery strategies for single cross-reactive T cell epitopes: traditional HCoV vaccines often contain complex full-length protein sequences or multiple antigens, making it difficult to achieve efficient targeted delivery of T cell epitopes, resulting in low efficiency of specific cellular immune responses and thus limiting the broad-spectrum protective effect of vaccines. Therefore, comprehensively analyzing the cross-reactive T cell epitope maps among HCoVs and developing broad-spectrum cross-reactive T cell epitope delivery technologies is undoubtedly the preferred strategy for addressing current and potential HCoV threats. Summary of the Invention

[0003] This invention provides a cross-reactive T-cell epitope map of human respiratory coronaviruses and its delivery method. First, this invention maps the cross-reactive T-cell epitopes among seven HCoVs, and further screens highly conserved epitope fragments from the identified cross-episodes, providing a core theoretical basis and experimental foundation for the development of broad-spectrum HCoV immune targets. Then, targeting the screened conserved epitope fragments, a precise delivery system for a Venezuelan equine encephalitis virus replicon particle (VRP) vaccine expressing a single conserved epitope is constructed, achieving targeted delivery of a "single conserved cross-reactive T-cell epitope," thus solving the key technical bottleneck of low cross-episode delivery efficiency in existing HCoV vaccines.

[0004] The first object of the present invention is to provide a coronavirus cross-reactive T-cell epitope peptide, which is a polypeptide of (a) or (b) or (c) below:

[0005] (a) A human respiratory coronavirus cross-reactive T-cell epitope peptide, the amino acid sequence of which is shown in any one of SEQ ID NO.1-SEQ ID NO.23;

[0006] (b) A polypeptide derived from (a) in which one or more amino acids have been substituted, deleted, or added to the amino acid sequence in (a) while retaining the cross-reactivity with coronaviruses;

[0007] (c) A highly conserved polypeptide in coronaviruses from different sources, the amino acid sequence of which is shown in any one of SEQ ID NO:24-SEQ ID NO:41, preferably, the highly conserved polypeptide is derived from nucleocapsid protein.

[0008] Preferably, the amino acid sequence of the human respiratory coronavirus cross-reactive T-cell epitope peptide described in item (a) is as shown in SEQ ID NO.13 or SEQ ID NO.14.

[0009] Preferably, the human respiratory coronaviruses described in (a) include SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, HCoV-OC43, HCoV-NL63 and HCoV-229E.

[0010] Preferably, the human respiratory coronavirus cross-reactive T cell epitope peptide described in item (a) is derived from human respiratory coronavirus structural proteins (S, N, M, E).

[0011] A second objective of this invention is to provide a nucleic acid sequence encoding the aforementioned coronavirus cross-reactive T-cell epitope peptide.

[0012] A third objective of this invention is to provide a recombinant vector comprising the aforementioned nucleic acid sequence encoding a coronavirus cross-reactive T-cell epitope peptide.

[0013] Preferably, the recombinant vector is a viral replicon particle.

[0014] More preferably, the viral replicon particle is a Venezuelan equine encephalitis virus replicon particle (VRP).

[0015] A fourth object of the present invention is to provide a host cell that can transduce or load the aforementioned coronavirus cross-reactive T-cell epitope peptide or the aforementioned nucleic acid sequence, or that can express / successfully encapsulate the aforementioned recombinant vector. Preferably, the host cell is a BHK-21 cell.

[0016] A fifth object of the present invention is to provide a vaccine in which the active ingredient is selected from any one of the following:

[0017] (1) The above-mentioned coronavirus cross-reactive T-cell epitope peptides;

[0018] (2) The above-mentioned nucleic acid sequence encoding the coronavirus cross-reactive T-cell epitope peptide;

[0019] (3) The above-mentioned recombinant vectors containing nucleic acid sequences;

[0020] (4) Host cells loaded with or transduced with the above-mentioned coronavirus cross-reactive T-cell epitope peptides.

[0021] A sixth object of the present invention is to provide an application comprising any one of the following:

[0022] (1) Application of the above epitope peptides in the preparation of coronavirus vaccines;

[0023] (2) Application of the above epitope peptides in the preparation of coronavirus cross-reactive T cells;

[0024] (3) Application of the above epitope peptides in the preparation of products for monitoring coronavirus evolution;

[0025] (4) Application of the above epitope peptides in the preparation of kits for inducing coronavirus cross-reactive T cells;

[0026] (5) Application of the above epitope peptides in the preparation of kits for detecting coronavirus infection;

[0027] (6) Application of the above nucleic acid sequences in the preparation of coronavirus vaccines;

[0028] (7) Application of the above-mentioned recombinant vectors in the preparation of coronavirus vaccines;

[0029] (8) Application of the above-mentioned host cells in the preparation of coronavirus vaccines.

[0030] Preferably, the coronavirus is a human respiratory coronavirus.

[0031] The seventh object of the present invention is to provide a passive immunotherapy agent against coronaviruses, the active ingredient of which is a coronavirus cross-reactive T cell, wherein the coronavirus cross-reactive T cell is obtained by stimulating lymphocytes with the above-mentioned coronavirus cross-reactive T cell epitope peptide or antigen-presenting cells loaded with the above-mentioned coronavirus cross-reactive T cell epitope peptide.

[0032] Preferably, the coronavirus is a human respiratory coronavirus, and the lymphocytes are C57BL / 6 mouse lymphocytes, more preferably BALF-derived cells.

[0033] An eighth object of the present invention is to provide a method for stimulating, initiating and / or amplifying T cells, comprising administering an effective amount of the above-described coronavirus cross-reactive T cell epitope peptide, the above-described nucleic acid sequence or the above-described recombinant vector; said method is for non-diagnostic or therapeutic purposes.

[0034] Preferably, the coronavirus is a human respiratory coronavirus, and the T cells are CD4⁺ T cells.

[0035] Beneficial effects:

[0036] This invention uses C57BL / 6 mice as a research platform, effectively overcoming the limitations of difficult clinical sample acquisition and significant individual variability, providing a stable and reproducible experimental basis for systematically analyzing the cross-reactive T-cell responses of seven HCoVs. Based on this, this invention clarifies for the first time the complete cross-reactivity network and cross-reactive T-cell epitope map among the seven HCoVs; simultaneously, it discovers conserved cross-reactive CD4⁺ T-cell epitopes from coronaviruses of different origins that target the N protein and possess multifunctional immunomodulatory activity. This not only breaks through the bottleneck of existing vaccine targets being limited to single viruses and lacking broad-spectrum protective potential, but also suggests that conserved cross-reactive epitopes are feasible targets for designing pan-coronavirus T-cell vaccines and elucidating their mechanisms of action, which can promote the development of broad-spectrum coronavirus vaccines. Furthermore, this invention successfully achieves the precise delivery of a single broad-spectrum cross-reactive CD4⁺ T-cell epitope. In summary, this invention provides key target resources for the development of broad-spectrum coronavirus T-cell vaccines and offers an application strategy for vaccine delivery of single conserved cross-reactive T-cell epitopes. Attached Figure Description

[0037] Figure 1These are the results of cross-reactive T-cell responses among the seven HCoVs in Example 1, where... Figure 1 Upper half: CD4 + T cell cross-response status; lower half: CD8 + T-cell cross-response.

[0038] Figure 2 It is the single cross-reactive CD4⁺ T cell epitope (MERS-CoV-N) expressed in Example 2. 98-112 The image shows the delivery effect of the VRP vaccine (VRP-MERS-N98).

[0039] Figure 3 It is the single cross-reactive CD4⁺ T cell epitope (SARS-CoV-2) expressed in Example 2. 109-123 The image shows the delivery effect of the VRP vaccine (VRP-SARS-N109). Detailed Implementation

[0040] The present invention will now be described in further detail with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the invention. The purpose of providing these embodiments and examples is to enable a more thorough and complete understanding of the disclosure of the present invention. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present invention, and the equivalent forms obtained also fall within the protection scope of the present invention. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for descriptive purposes only and is not intended to be limiting of the invention.

[0042] the term

[0043] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0044] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0045] In this invention, terms such as "further" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this invention.

[0046] Moreover, terms such as "first," "second," "third," and "fourth" serve only as non-exhaustive enumerations and should be understood as not constituting a closed limitation on the quantity.

[0047] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0048] All references to this invention are incorporated herein by reference as if each document were individually incorporated by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, the referenced documents involved in this invention are incorporated in their entirety and for all purposes. When references are made in this invention, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When references are made in this invention, examples and preferred embodiments of the relevant technical features cited may also be incorporated herein by reference, but only to the extent that they enable the implementation of this invention. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptively based on the description in this application.

[0049] As used in this article, the term "T-cell epitope" refers to a short peptide formed after an antigen is processed by the proteasome, which needs to bind to an MHC molecule and be presented on the cell surface, where it is specifically recognized by the T-cell receptor (TCR), thereby mediating an immune response from CD4⁺ or CD8⁺ T cells. "Cross-epitope" refers to an epitope present in different antigens (such as different viruses, bacteria, or proteins), possessing structural or sequence similarity, and being specifically recognized by the same immune receptor (such as TCR or antibody) to trigger a cross-immune response. "Conservation" in molecular biology and virology refers to the characteristic of key sequences or structures of biological macromolecules (such as nucleic acids or proteins) exhibiting low frequency and degree of variation during long-term evolution, resulting in high retention across different species or strains. "Cross-reactivity" in immunology refers to the phenomenon where the immune system's response to a particular antigen (such as T / B cell activation) can simultaneously recognize and act on that heterologous antigen due to its structural similarity to another antigen from a different source. "Broad-spectrum" in the context of this study refers to the broad recognition ability of T cells for similar antigens or epitopes in different coronaviruses.

[0050] The following are some specific examples.

[0051] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following examples should preferably refer to the guidelines given in this invention, or be performed according to experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturer, or refer to experimental methods known in the art. In some embodiments of the present invention, the host cells used in the entire experimental system are CHB3 cells, BHK-21 cells, AD293 cells, Huh7-ACE2 cells, Huh7 cells, VeroE6 cells, or Vero81 cells; the T cell epitope expression vector is a VRP vaccine vector; the HCoV receptor expression vector is an adenovirus vector; and the experimental animal model is a C57BL / 6 mouse.

[0052] Example 1: Conserved sequence analysis of CD4⁺ T cell epitopes using BlastP

[0053] We provide cross-reactive human respiratory coronavirus T-cell epitope peptides and their corresponding amino acid sequences, thereby identifying conserved sequences located on the N protein of various coronaviruses.

[0054] I. Steps for constructing a cross-reactive T cell epitope map

[0055] A step-by-step research strategy was adopted to identify cross-reactive T cell epitopes using intracellular cytokine staining (ICS) combined with flow cytometry. The specific steps are as follows:

[0056] The first step involved stimulating mice with bronchoalveolar lavage fluid (BALF) cells after infection or immunization with a total peptide library formed by mixing peptides of four structural proteins of different human respiratory coronaviruses (HCoVs). The expression of cytokine IFN-γ was detected by ICS combined with flow cytometry. Using No pep + 3SD as the criterion, total peptide libraries that could induce the expression of IFN-γ were screened and defined as cross-reactive coronaviruses and cross-reactive coronavirus total peptide libraries.

[0057] The second step involved immunizing mice with a Venezuelan equine encephalitis virus replicon vaccine (VRP) expressing a single coronavirus structural protein. Subsequently, BALF-derived cells were stimulated with a single structural protein peptide library of cross-reactive coronaviruses. By detecting IFN-γ expression, it was determined whether specific T cells targeting a particular coronavirus structural protein could cross-recognize the structural protein of a cross-reactive coronavirus, thereby identifying the target protein that induces cross-reactivity.

[0058] The third step involves comparing the sequence conservation of the target coronavirus with that of other coronaviruses through bioinformatics analysis, combining the conserved regions of known virus-specific T-cell epitopes, synthesizing sequence-similar peptides (as cross-reactive epitope candidates) in the corresponding structural proteins of cross-reactive coronaviruses, and then verifying them experimentally using ICS combined with flow cytometry.

[0059] Taking the identification of cross-reactive T-cell epitopes of SARS-CoV-2 as an example, the specific implementation is as follows:

[0060] 1. Using peptide libraries of structural proteins from seven human respiratory coronaviruses, BALF cells of mice infected with SARS-CoV-2 were stimulated. Using IFN-γ as a response indicator and No pep + 3SD as the criterion, it was identified that SARS-CoV-2 specific T cells could cross-recognize the total SARS-CoV peptide library.

[0061] 2. Mice were immunized with VRPs expressing single structural proteins (S, N, M, E) of SARS-CoV-2. Subsequently, BALF-derived cells after VRP immunization were stimulated with peptide libraries of single structural proteins S, N, M, and E of SARS-CoV. By detecting IFN-γ expression, it was confirmed that SARS-CoV-2 S, N, and M specific T cells can cross-recognize homologous structural proteins of SARS-CoV.

[0062] 3. Based on the known SARS-CoV-2 T cell epitopes, the conservation of structural protein sequences of SARS-CoV-2 and SARS-CoV was analyzed by sequence alignment. Peptides with similar sequences were synthesized from the corresponding structural proteins of SARS-CoV and verified by ICS combined with flow cytometry.

[0063] The results of cross-reactive T-cell responses among the seven HCoVs are as follows: Figure 1 As shown in Table 1, the complete epitope map and corresponding amino acid sequences are shown in Table 1.

[0064] Table 1. Summary of cross-reactive T-cell epitopes of 7 HCoVs ; ;

[0065] II. Conservative Analysis

[0066] Based on the above map, the cross-reactive CD4⁺ T epitope MERS-CoV-N was confirmed through sequence alignment. 98-112 (SEQ ID NO.13) and SARS-CoV-N 109-123 The conservation of (SEQ ID NO.14) within the coronavirus genus is demonstrated through the following steps:

[0067] a) Sequence acquisition: Amino acid sequences of target cross-epitopes, including MERS-CoV-N, were extracted from the plotted cross-reactive T cell epitope map (Table 1). 98-112 (The sequence is shown in SEQ ID NO.13: WYFYYTGTGPEAALP) and SARS-CoV-N 109-123 (The sequence is shown in SEQ ID NO.14: WYFYYLGTGPEASLP).

[0068] b) Conservation analysis: The protein-protein BLAST (BlastP) sequence alignment tool was used to align the two target sequences with different coronavirus strains to analyze the degree of conservation of the epitope in different strains and screen out cross-epitopes or sequences with high conservation, as shown in Table 2.

[0069] Table 2. Summary of conserved sequences of coronaviruses from different sources ; ;

[0070] Example 2: Achieving efficient delivery of a single broad-spectrum cross-reactive CD4⁺ T cell epitope

[0071] Based on the conserved sequences identified in Example 1, a technical platform was developed to achieve precise delivery of a single broad-spectrum cross-reactive T cell epitope. To achieve this objective, the following experimental system was constructed: the host cells used were CHB3 cells and BHK-21 cells; the expression vector was a VRP vaccine vector; and the experimental animal model was a C57BL / 6 mouse.

[0072] 1. Preparation of core experimental materials: Construction of a single-epitope VRP vaccine

[0073] Using gene cloning technology, the highly conserved cross-epitope sequences identified in Example 1 were inserted into the Venezuelan Equine Encephalitis virus replicon particle (VRP) vaccine vector to construct a VRP vaccine expressing only a single CD4⁺ T cell epitope, as follows: Vaccine 1: MERS-CoV-N 98-112 The epitope sequence (WYFYYTGTGPEAALP) was cloned into the VRP vector and named VRP-MERS-CoV-N98-112, abbreviated as VRP-MERS-N98; Vaccine 2: SARS-CoV-N 109-123 The epitope sequence (WYFYYLGTGPEASLP) was cloned into the VRP vector and named VRP-SARS-CoV-N109-123, or VRP-SARS-N109 for short.

[0074] The specific construction process of the above VRP vector (such as plasmid construction and synthesis, enzyme digestion, in vitro transcription, purification and identification) was performed in accordance with the methods in existing literature (Pushko, P.). et al. Replicon-helper systems from attenuatedVenezuelan equine encephalitis virus: expression of heterologous genes invitro and immunization against heterologous pathogens in vivo. Virology 239,389-401 (1997). https: / / doi.org:10.1006 / viro.1997.8878), ensuring that the vaccine can efficiently express the target epitope antigen.

[0075] 2. Verification of VRP vaccine immunization efficacy

[0076] The VRP vaccine was administered intranasally to female C57BL / 6 mice, approximately 6 weeks old. BALF was collected on day 7 and administered MERS-CoV-N... 98-112 (Vaccine 1 Immunization) and SARS-CoV-2 109-123 (Vaccine 2 Immunization) The peptide was used as a stimulant for in vitro stimulation, and the virus-specific T cell response was detected by intracellular cytokine staining and flow cytometry.

[0077] The steps are briefly described as follows:

[0078] 2.1 VRP vaccine intranasal immunization

[0079] Anesthetize the mice: Add an appropriate amount of isoflurane to the anesthesia container and place the mice in it. Observe the mice's breathing: from slowing down to starting to speed up, and then slowing down again to deep abdominal breathing and maintaining a steady state, remove the mice. Keep the mice upright, i.e., with their heads and bodies in the same vertical line, and use a 200 µL pipette to draw 50 µL of diluted VRP and drip it into the mouse's nose (VRP vaccine immunization dose: 1E5 IU). After dripping into the nose, keep the mice in an upright position for 5-10 seconds before returning them to their cages.

[0080] 2.2 Collection of bronchoalveolar lavage fluid and preparation of single-cell suspension in mice

[0081] a) Mouse anesthesia: Avertin (2,2,2-tribromoethanol) was injected intraperitoneally into mice at a dose of 350 µL / 20g, and the mice were anesthetized in about 2 minutes.

[0082] b) Mouse fixation: Place the mouse supine on a mat covered with surgical cloth and fix its limbs and nose tip.

[0083] c) Disinfect the fur around the neck with 75% alcohol, expose the trachea and cut it open.

[0084] d) Use a 1 mL syringe containing RPMI 1640 medium with 5% or 10% fetal bovine serum to draw bronchoalveolar lavage fluid.

[0085] e) Centrifuge at 400 g and 4 ℃ for 5 min. Discard the liquid using a vacuum pump and resuspend it in RP10 medium (RPMI 1640 medium containing 10% fetal bovine serum) to obtain a single-cell suspension of mouse bronchoalveolar lavage fluid.

[0086] 2.3 In vitro stimulation and intracellular cytokine staining

[0087] Virus-specific T-cell responses were detected using in vitro stimulation and intracellular cytokine staining. Stimulation of lymphocytes derived from bronchoalveolar lavage fluid required antigen-presenting cells CHB3 and CD4. +T-cell polypeptide (i.e., the aforementioned MERS-CoV-N) 98-112 and SARS-CoV-N 109-123 The peptide was used at a final concentration of 5 μM. The specific steps are as follows:

[0088] a) Label 96-well circular plates according to the experimental purpose, and equilibrate the Golgi transport inhibitor (BFA) to room temperature.

[0089] b) Aspirate 100 µL / well of the resuspended mouse bronchoalveolar lavage fluid single-cell suspension into a 96-well round-bottom plate.

[0090] c) Dilute BFA with RP10 medium to a final concentration of 1:1000.

[0091] d) Peptide preparation: First, resuspend CHB3 cells in RP10 medium containing BFA prepared in step c) until the cell concentration reaches 5 × 10⁻⁶ cells / cells. 6 -1 × 10 7 / mL, then prepare a 2× peptide and CHB3 cell mixture, adding it to the reaction system at a volume of 100 µL / well; the control setting uses a reaction system without the target peptide: resuspend CHB3 cells in RP10 medium containing only BFA (without peptide), adjusting the cell concentration to 5 × 10⁶. 6 -1 × 10 7 Add 100 µL / well to the reaction system at a volume of 1 mL / well.

[0092] e) Add 2 × peptide mixture to a 96-well round-bottom plate and mix thoroughly with the single-cell suspension. Incubate at 37 ℃ in a 5% CO2 cell culture incubator for 6 h.

[0093] f) Centrifuge at 400 g and 4 ℃ for 5 min, then discard the supernatant.

[0094] g) Surface antibody staining: Prepare surface antibody using FACS buffer, 100 µL / well. After thorough mixing, stain at 4 °C in the dark for 15 min.

[0095] h) Wash cells: Add FACS Buffer, 120 µL / well. Centrifuge at 400 g, 4 ℃ for 5 min, and discard the supernatant.

[0096] i) Fixation and membrane disruption: Add BD Cytofix / Cytoperm, 135 µL / well. Mix thoroughly and stain at 4 °C in the dark for 30 min.

[0097] j) Wash cells: Add 1 × Perm / Wash, 120 µL / well. Centrifuge at 800 g, 4 ℃ for 5 min, and discard the supernatant.

[0098] k) Intracellular staining: Prepare intracellular antibody using 1 × Perm / Wash, 100 µL / well. After thorough mixing, stain at 4 ℃ in the dark for 30 min.

[0099] 1) Wash cells: Add 1 × Perm / Wash, 120 µL / well. Centrifuge at 800 g, 4 ℃ for 5 min, and discard the supernatant.

[0100] m) Resuspend: Add FACS Buffer, 100 µL / well. Mix thoroughly and transfer cells to flow cytometry tubes.

[0101] n) Samples were analyzed using a BD FACS Verse flow cytometer and data were processed using FlowJo professional analysis software.

[0102] Experimental results: Results of VRP-MERS-N98 immunization in mice are shown in [the table below]. Figure 2 VRP-MERS-N98 immunization can successfully induce recognition of MERS-CoV-N 98-112 Peptide-specific T cells; results of VRP-SARS-N109 immunization in mice are shown in [see image]. Figure 3 VRP-SARS-N109 immunization can successfully induce the recognition of SARS-CoV-N 109-123 Peptide-specific T cells.

Claims

1. A coronavirus cross-reactive T-cell epitope peptide, characterized in that, The polypeptide is one of the following (a), (b), or (c): (a) A human respiratory coronavirus cross-reactive T-cell epitope peptide, the amino acid sequence of which is shown in any one of SEQ ID NO.1-SEQ ID NO.23; (b) A polypeptide derived from (a) in which one or more amino acids have been substituted, deleted, or added to the amino acid sequence in (a) while retaining the cross-reactivity with coronaviruses; (c) A polypeptide that is highly conserved in coronaviruses from different sources, having an amino acid sequence as shown in any one of SEQ ID NO: 24-SEQ ID NO:

41.

2. The coronavirus cross-reactive T-cell epitope peptide according to claim 1, characterized in that, The amino acid sequence of the human respiratory coronavirus cross-reactive T-cell epitope peptide described in item (a) is shown in SEQ ID NO.13 or SEQ ID NO.

14.

3. The coronavirus cross-reactive T-cell epitope peptide according to claim 1 or 2, characterized in that, The human respiratory coronaviruses mentioned in (a) include SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, HCoV-OC43, HCoV-NL63 and HCoV-229E.

4. A nucleic acid sequence, characterized in that, It encodes the coronavirus cross-reactive T-cell epitope peptide as described in claim 1, 2 or 3.

5. A recombinant vector, characterized in that, It comprises the nucleic acid sequence of claim 4; preferably, the recombinant vector is a Venezuelan equine encephalitis virus replicon particle.

6. A host cell, characterized in that, It can transduce or load the epitope peptide of claim 1, 2 or 3 or the nucleic acid sequence of claim 4, or can express / successfully encapsulate the recombinant vector of claim 5; preferably, the host cell is a BHK-21 cell.

7. A vaccine, characterized in that, Its active ingredient is selected from any of the following: (1) The coronavirus cross-reactive T-cell epitope peptide according to claim 1, 2 or 3; (2) The nucleic acid sequence according to claim 4; (3) The recombinant vector according to claim 5; (4) Loading or transducing the host cell with the coronavirus cross-reactive T cell epitope peptide of claim 1, 2 or 3.

8. An application characterized in that, The application includes any of the following: (1) The use of the epitope peptide according to claim 1, 2 or 3 in the preparation of coronavirus vaccines; (2) The use of the epitope peptide according to claim 1, 2 or 3 in the preparation of coronavirus cross-reactive T cells; (3) The use of the epitope peptide according to claim 1, 2 or 3 in the preparation of products for monitoring the evolution of coronaviruses; (4) The use of the epitope peptide according to claim 1, 2 or 3 in the preparation of a kit for inducing coronavirus cross-reactive T cells; (5) The use of the epitope peptide according to claim 1, 2 or 3 in the preparation of a kit for detecting coronavirus infection; (6) The use of the nucleic acid sequence of claim 4 in the preparation of a coronavirus vaccine; (7) The use of the recombinant vector according to claim 5 in the preparation of a coronavirus vaccine; (8) The use of the host cell as described in claim 6 in the preparation of a coronavirus vaccine; Preferably, the coronavirus is a human respiratory coronavirus.

9. A passive immunotherapy agent against coronaviruses, characterized in that, Its active ingredient is coronavirus cross-reactive T cells, which are obtained by stimulating lymphocytes with the coronavirus cross-reactive T cell epitope peptide as described in claim 1, 2 or 3 or antigen-presenting cells loaded with the coronavirus cross-reactive T cell epitope peptide as described in claim 1, 2 or 3. Preferably, the coronavirus is a human respiratory coronavirus and the lymphocytes are C57BL / 6 mouse-derived lymphocytes.

10. A method for stimulating, initiating, and / or expanding T cells, characterized in that, It includes administering an effective amount of the coronavirus cross-reactive T-cell epitope peptide of claim 1, 2 or 3, the nucleic acid sequence of claim 4 or the recombinant vector of claim 5; the method is for non-diagnostic or therapeutic purposes; preferably, the coronavirus is a human respiratory coronavirus and the T cells are CD4⁺ T cells.

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