HLA-E restrictive SARS-CoV-2 specific CD8 + T cell epitope peptide as well as polymer and application thereof

Abstract

The invention belongs to the technical field of prevention and treatment of severely acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), and discloses an HLA-E restrictive SARS-CoV-2 specific CD8 + T cell epitope peptide, a polymer of the HLA-E restrictive SARS-CoV-2 specific CD8 + T cell epitope peptide and application of the HLA-E restrictive SARS-CoV-2 specific CD8 + T cell epitope peptide, and amino acid sequences of the HLA-E restrictive SARS-CoV-2 specific CD8 + T cell epitope peptide are shown as SEQ ID NO: 1-2. The HLA-E restricted CD8 < + > T cell epitope polypeptide provided by the invention can be applied to preparation of epitope peptide vaccines or induction of generation of epitope peptide specific CTL, so that the problem of low population coverage rate caused by HLA restriction of the existing vaccines is effectively solved, immune response coverage of wider crowds is realized through non-classical HLA-E molecules, and the HLA-E restricted CD8 < + > T cell epitope polypeptide can be applied to preparation of epitope peptide vaccines. The traditional Chinese medicine composition has the advantage of improving the universal immune response of people.

Description

Technical Field

[0001] This invention belongs to the field of prevention and treatment technology for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and relates to HLA-E-restricted SARS-CoV-2-specific CD8. + T-cell epitope peptides, their polymers, and applications. Background Technology

[0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a beta coronavirus, a single-stranded positive-sense enveloped RNA virus. Its genome is approximately 29.8 kb in size. The two open reading frames (ORFs) at the 5' end, ORF1a and ORF1b, encode non-structural proteins (nsp). The 3' end encodes structural proteins: spike protein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N), as well as eight accessory proteins (ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8b, ORF9b, and ORF10). The S protein recognizes the angiotensin-converting enzyme 2 (ACE2) receptor on host cells via its receptor-binding domain (RBD), mediating viral entry into host cells. The M protein is structurally abundant and sequence-conserved, mediating viral assembly and budding, and maintaining viral morphogenesis. The E protein has a small molecular weight and can form ion channels, assisting other proteins in completing viral assembly and budding. The N protein typically binds to viral nucleic acid material and plays an important role in viral capsid formation. All four structural proteins possess varying degrees of immunogenicity and are important sources of antigenic epitopes. The S protein has strong immunogenicity and is an important protein for effectively inducing humoral and cellular immune responses. Its RBD and specific epitopes are core targets for the development of therapeutic neutralizing antibodies and the study of specific T cell response mechanisms. The N protein has an immunodominant epitope at its C-terminus, which can induce specific T cell responses. The M protein contains immunodominant epitopes at its N-terminus, C-terminus, and transmembrane domain, which can induce protective humoral immune responses and strong T cell responses. Although non-structural proteins and some accessory proteins have low protein content in infected cells, they also contain the very important CD8+. + T-cell epitopes, such as ORF9b-derived epitopes, can induce high levels of CD8 in COVID-19 recovered individuals. + T-cell response confirmed its good immunogenicity.

[0003] Numerous studies have shown that during SARS-CoV-2 infection, CD8... + T cells can recognize viral antigens presented by major histocompatibility complex (MHC) class I molecules on antigen-presenting cells (APCs) through their T cell receptors (TCRs). They then kill target cells by secreting granzymes and perforin, or induce programmed cell death in target cells through death receptor-ligand interactions (such as Fas-FasL), thereby effectively clearing virus-infected cells and limiting viral replication and spread. Therefore, CD8... + T cells play a crucial role in the body's immune response to viral infections and are essential for the generation and maintenance of long-term immune memory after infection. Identification of SARS-CoV-2 specific CD8 cells... + T-cell epitopes are a key area for in-depth exploration of specific CD8+. + The important foundation for the function and mechanism of T cells, currently targeting SARS-CoV-2 specific CD8 + Studies on T cell epitopes have mostly been limited to CD8 restriction by classic HLA class I molecules, such as HLA-A*O2 and HLA-B*O7. + T-cell epitopes. It is noteworthy that non-classical HLA-I molecules can also present antigenic epitopes for specific CD8. + T cell recognition, due to the low polymorphism of its alleles, can be used as a tool for studying CD8. + T cell responses in the general population and the preferred molecules for designing more specific and effective peptide vaccines. However, to date, there is limited information on non-classical HLA-I molecules restricting SARS-CoV-2-specific CD8. + The identification and research reports on T-cell epitopes are very limited. This situation restricts our understanding of the adaptive immune response patterns after SARS-CoV-2 infection and also limits progress in areas such as peptide vaccine development, disease treatment strategy formulation, and optimization of epidemic control measures. Non-classical HLA-I molecules mainly include HLA-E, HLA-G, and HLA-F, among which HLA-E is the most well-studied non-classical HLA-I molecule. HLA-E has a similar structure to classical HLA-I molecules, consisting of a heavy chain (α chain) and β2 microglobulin (β2m). The α1 and α2 domains at the distal membrane end of the extracellular region of its heavy chain form an antigen-binding groove. HLA-E molecules are widely expressed in vivo, but due to the lack of high-affinity endogenous peptides to bind to it, HLA-E expression on the cell surface is unstable.

[0004] Vaccination is currently the most effective way to prevent SARS-CoV-2 infection. Widespread vaccination against SARS-CoV-2 has significantly reduced both the infection rate and the rate of severe illness. However, the repeated mutations of the SARS-CoV-2 strain have posed a significant challenge to epidemic prevention and control and medical treatment, severely impacting the effective prevention and treatment of COVID-19. Currently, the SARS-CoV-2 vaccines that have been developed and marketed mainly include inactivated virus vaccines, recombinant protein vaccines, and adenovirus vector vaccines. These vaccines can effectively induce the production of neutralizing antibodies and provide significant protection against severe illness and death. However, the neutralizing antibody titers of inactivated and recombinant protein vaccines decrease significantly over time, and their neutralizing ability against highly variant strains is limited; while adenovirus vector vaccines can induce strong CD8+... + T-cell responses are effective, but humoral immunity is relatively weak and it is difficult to build a long-lasting immune barrier. Therefore, further research and development of more effective new vaccines are needed.

[0005] In recent years, synthetic peptide vaccines have become an important direction in novel vaccine development due to their precise targeting of immune epitopes. Their core advantage lies in the fact that the synthesized short peptides can directly bind to HLA-I molecules without intracellular processing by APCs, and through optimized design, they can mimic the natural endogenous peptide-HLA-I complex, efficiently activating CD8. + T cells. Currently, peptide vaccines targeting HPV, HIV, and melanoma have entered clinical trials. Based on CD8. + Synthetic peptide vaccines based on T-cell epitopes primarily induce specific cellular immune responses, offering advantages such as no risk of genomic integration, high stability, and short development cycles. Furthermore, by combining conserved epitopes from multiple pathogens, broad-spectrum, multivalent vaccines can be designed, providing an optimized strategy for addressing pathogen mutations. In SARS-CoV-2 research, it was found that T-cell epitopes are mostly concentrated in conserved regions of the SARS-CoV-2 genome. Although single-point mutations in some variants may lead to escape responses in individual T-cell clones, the diversity of the T-cell receptor repertoire and epitope cross-reactivity ensure that specific CD8+ epitopes remain highly effective. + T-cell responses still maintain a high level of recognition ability against variant strains. The development of peptide vaccines based on SARS-CoV-2 T-cell epitopes relies on the accurate identification of highly conserved and strongly immunogenic CD8+. + T-cell epitopes need to be explored, while also overcoming the problem of low HLA-restricted response coverage in the population.

[0006] In summary, current SARS-CoV-2 vaccine development primarily relies on classical HLA-I molecular restriction CD8 receptors. +T cell epitopes, but due to the high polymorphism of classical HLA alleles, the coverage of population immune responses is limited. Current epitope screening strategies focus on classical molecules such as HLA-A and HLA-B, and particularly lack targeting of HLA-E-restricted CD8+. + Systematic studies of T-cell epitopes have shown that the HLA-E molecule has only two functional alleles, and the allele distribution is even in the Chinese population. However, the lack of systematic studies on HLA-E-restricted SARS-CoV-2 epitopes has hindered the development of broad-spectrum vaccines based on non-classical HLA molecules, which has limited the progress of developing novel broad-spectrum vaccines. Summary of the Invention

[0007] To overcome the shortcomings of the prior art, the present invention aims to provide a class of HLA-E-restricted SARS-CoV-2 protective CD8 receptors. + T-cell epitope peptides and their applications to address existing vaccine targets for CD8 + The problem lies in the weak T-cell immune response and the low coverage of induced responses in the population.

[0008] To achieve the above objectives, the present invention employs the following technical solution: This invention discloses a class of HLA-E-restricted SARS-CoV-2 specific CD8 receptors. + T-cell epitope peptides, after binding to and being presented with HLA-E molecules, can induce CD8+. + T cells generate cellular immune responses.

[0009] Preferably, the epitope peptide binds complementaryly to the antigen-binding groove formed by the α1 and α2 domains of the HLA-E molecule; during the binding process, the hydrophobic residues of the 2nd and 9th amino acids of the epitope peptide are embedded in the anchoring sites of the HLA-E molecule binding groove, while the polar residues in the middle region form a hydrogen bond network with the HLA-E molecule, forming a stable complex structure.

[0010] Preferably, the amino acid sequence of the epitope peptide is as shown in SEQ ID NO:1 or SEQ ID NO:2.

[0011] This invention also discloses the above-mentioned HLA-E-restricted SARS-CoV-2 specific CD8. + Application of T-cell epitope peptides in the preparation of SARS-CoV-2 epitope peptide vaccines.

[0012] Preferably, the SARS-CoV-2 epitope peptide vaccine is capable of inducing the production of epitope peptide-specific CD8+. + T-cell epitope peptide vaccine.

[0013] This invention also discloses an HLA-E / epitope peptide complex monomer, which is composed of the above-mentioned HLA-E-restricted SARS-CoV-2 specific CD8. + The complex is composed of T-cell epitope peptides and a complex monomer; the complex monomer is a structure assembled in vitro from an HLA-E molecular heavy chain with an activated biotin molecule linked to its C-terminus and β2 microglobulin.

[0014] The present invention also discloses a SARS-CoV-2 specific HLA-E / epitope peptide-complex tetramer, which is composed of an avidin molecule and four biotin-labeled HLA-E / epitope peptide complex monomers. The HLA-E / epitope peptide complex monomer is the HLA-E / epitope peptide complex monomer described above.

[0015] Preferably, the avidin molecule is labeled with fluorescein PE.

[0016] Preferably, the HLA-E / epitope peptide-complex tetramer can bind to epitope-specific CD8. + TCRs bind to the surface of T cells.

[0017] This invention also discloses the above-mentioned SARS-CoV-2 specific HLA-E / epitope peptide-complex tetramer in detecting SARS-CoV-2 epitope peptide-specific CD8+. + Applications in T cell frequency.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides HLA-E molecule-restricted SARS-CoV-2 specific CD8 + T-cell epitope peptides were obtained through epitope prediction software, HLA-E*0103 and HLA-E*0101 transfection of K562 cell lines, and ELISpot assays. The SARS-CoV-2 epitope peptides were experimentally confirmed to possess high specificity and can induce specific CD8+. + T cells generate a strong cellular immune response, secreting IFN-γ. The frequency of IFN-γ secretion can be used to indicate the degree of immune response of SARS-CoV-2 epitope peptide-specific CTLs; that is, the higher the frequency of IFN-γ secretion, the stronger the CD8+ of COVID-19 convalescent cells. + The stronger the T-cell immune response.

[0019] The SARS-CoV-2-specific CD8 receptor antagonist obtained by this invention is HLA-E restricted. +T-cell epitope peptides can be used in the preparation of epitope peptide vaccines, inducing epitope peptide-specific CTL responses, or preparing HLA-E / epitope peptide-complex tetramers. This provides important reference for designing more specific and effective peptide vaccines for the Chinese population and offers a powerful tool for assessing the establishment of immune barriers in recovered SARS-CoV-2 patients and evaluating the protective effect of SARS-CoV-2 vaccination. It also shows promising development and application prospects in the field of COVID-19 specific immunotherapy. Therefore, the HLA-E-restricted CD8+ peptides provided in this invention... + T-cell epitope peptides can be used in the preparation of epitope peptide vaccines or to induce epitope peptide-specific CTLs, effectively solving the problem of low population coverage caused by HLA restriction in existing vaccines. By using non-classical HLA-E molecules, they achieve broader immune response coverage in the population, thus improving the universality of the immune response. This invention also discloses CD8 restricted by HLA-E molecules. + T-cell epitope peptides were used to prepare HLA-E / epitope peptide-complex tetramers, which consist of four biotin-labeled HLA-E / epitope peptide-complex monomers bound to one avidin molecule. The four monomers are oriented to form a spatially multivalent structure, which enhances the binding stability of the tetramer to T-cell surface TCRs and improves detection sensitivity. Therefore, the prepared tetramer can be used to prepare a assay for detecting SARS-CoV-2 epitope peptide-specific CD8+. + Reagents for T cell frequency.

[0020] Furthermore, the avidin molecule in the tetramer structure of the present invention is labeled with fluorescein PE. The PE labeling strategy has irreplaceable advantages in detecting rare T cell populations. For example, the high brightness and low background noise characteristics of PE can significantly improve the signal-to-noise ratio when detecting antigen-specific T cells at extremely low frequencies, thus avoiding false negative results. Attached Figure Description

[0021] Figure 1 To screen K562 cells for HLA-E*0103 or HLA-E*0101 transfection, binding assays were performed to screen for high-affinity SARS-CoV-2-specific CD8+ of HLA-E*0103 or HLA-E*0101. + Figure 1 shows the results of T cell 9-peptide epitope transfection; where A represents K562 cells transfected with HLA-E*0103; and B represents K562 cells transfected with HLA-E*0101. Figure 2A graph representing the frequency of IFN-γ secretion by HLA-E high-affinity SARS-CoV-2 9-peptide-specific CTLs in peripheral blood of SARS-CoV-2 convalescent patients detected by ELISpot (using the test results of COVID-19 convalescent patient No. 243 as an example); Figure 3 This figure shows the comparison of IFN-γ secretion frequency by HLA-E-restricted SARS-CoV-2 epitope-specific CTLs between the COVID-19 convalescent population and healthy controls (HC) (PBMCs specimens frozen in our laboratory before 2020 that were not infected with SARS-CoV-2); where A is the VMVELVAEL result figure; B is the SMQNCVLKL result figure; Figure 4 A representative graph showing the frequency of HLA-E-restricted SARS-CoV-2 epitope-specific CTLs in peripheral blood of SARS-CoV-2 convalescent patients detected by HLA-E / peptide-tetramer staining (using the results of COVID-19 convalescent patient No. 303 as an example). Figure 5 This figure shows the comparison of the frequencies of HLA-E-restricted SARS-CoV-2 9-peptide epitope-specific CTLs in the peripheral blood of different groups of COVID-19 convalescents and uninfected healthy controls. In the figures, A represents the comparison between the convalescent population and uninfected healthy controls; B represents the comparison between COVID-19 convalescents with different disease levels; C represents the comparison between symptomatic and asymptomatic COVID-19 convalescents; and D represents the comparison between COVID-19 convalescents who received 2 or 3 doses of SARS-CoV-2 vaccine. Detailed Implementation

[0022] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0023] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0024] The present invention will now be described in further detail with reference to the accompanying drawings: In existing technologies, SARS-CoV-2 vaccine development mainly relies on classical HLA-I molecular restriction CD8. + T-cell epitopes, however, are limited in their coverage by the population immune response due to the high polymorphism of classical HLA alleles. Current epitope screening strategies focus on classical molecules such as HLA-A and HLA-B, while non-classical molecules such as HLA-E have not been fully explored due to their low polymorphism. The HLA-E molecule has only two functional alleles, and the allele distribution is even in the Chinese population. However, current technologies lack systematic research on HLA-E-restricted SARS-CoV-2 epitopes, hindering the development of broad-spectrum vaccines based on non-classical HLA molecules.

[0025] The HLA-E gene locus is highly conserved in the population, with two main alleles, HLA-E*0101 and HLA-E*0103. In the Han Chinese population, the expression frequency of HLA-E*0103 is slightly higher than that of HLA-E*0101, and together they cover more than 99% of individuals. Furthermore, both alleles often present the same or similar epitope peptides, making HLA-E-based immunization strategies widely applicable. Therefore, based on the unique advantages of HLA-E molecules in antigen presentation—their low polymorphism can overcome the limitations of traditional HLA restriction on population coverage—this research focuses on screening viral-derived peptides that can stably bind to HLA-E. By analyzing the amino acid sequences of SARS-CoV-2 structural and non-structural proteins, combined with the spatial conformational characteristics of the HLA-E antigen-binding groove, candidate peptides adapted to its binding motif were screened. Further in vitro binding experiments were used to verify the affinity of the candidate peptides for HLA-E molecules, ultimately identifying the epitope sequences with stable binding ability.

[0026] Example 1 This invention proposes a class of HLA-E-restricted SARS-CoV-2-specific CD8 receptors.+ T-cell epitope peptides, whose amino acid sequences are shown in SEQ ID NO: 1-2.

[0027] HLA-E restriction refers to the specific binding ability of epitope peptides to form complexes with HLA-E molecules. This can be verified by in vitro peptide-HLA binding experiments, which are confirmed by measuring the binding stability of fluorescently labeled peptides to HLA-E molecules.

[0028] Among them, the amino acid sequence of epitope peptides refers to specific peptide segments screened from SARS-CoV-2 viral proteins. Specifically, bioinformatics can be used to predict the motif characteristics of binding HLA-E antigen binding grooves, such as a 9-peptide structure containing the conserved VMAPRTL / VV / L / FL sequence.

[0029] CD8 + T-cell epitope peptides refer to those that can be detected by CD8. + The antigenic determinants recognized by TCRs on the surface of T cells can be specifically verified through in vitro T cell activation experiments, such as detecting CD8+ after peptide stimulation. + The level of IFN-γ secreted by T cells.

[0030] Specifically, the polypeptide sequences shown in SEQ ID NO:1 and SEQ ID NO:2, through their specific amino acid residue arrangement, complementarily bind to the antigen-binding groove formed by the α1 and α2 domains of the HLA-E molecule. During binding, the hydrophobic residues at positions 2 and 9 (C-terminus) of the polypeptide embed into the anchoring sites of the HLA-E molecule's binding groove, while the polar residues in the middle region form a hydrogen bond network with the HLA-E molecule, thereby forming a stable complex structure. After this complex is transported to the cell surface, it can activate CD8 through a dual recognition mechanism of TCR, epitope peptide, and MHC. + T cells induce specific cytotoxic responses. Compared to classic HLA-restricted epitopes, this approach utilizes the low polymorphism of HLA-E to achieve higher response coverage in the population.

[0031] More specifically, the epitope peptides shown in SEQ ID NO: 1 and SEQ ID NO: 2 form a stable complex with the antigen-binding groove of the HLA-E molecule via their specific amino acid sequences. After being transported to the cell surface, this complex binds to CD8+. + The specific binding of TCRs on the surface of T cells triggers the T cell activation signaling pathway. Activated CD8 +T cells further differentiate into effector cells, secreting cytotoxic mediators such as granzymes and perforin, and releasing cytokines such as IFN-γ, thereby clearing virus-infected host cells. Because HLA-E molecules exhibit low polymorphism in the population, the aforementioned epitope peptides do not require peptide design targeting different HLA-I alleles and can induce immune responses in most individuals.

[0032] It can be seen that the existing CD8 + T-cell epitope screening primarily targets classic molecules such as HLA-A and HLA-B, whose allele polymorphisms lead to insufficient vaccine coverage. This invention, however, screens for HLA-E restrictive epitopes, leveraging their unique characteristic of having only two functional alleles, allowing the same epitope to cover carriers of different HLA-E alleles. Furthermore, HLA-E molecules exhibit a conservation advantage in viral antigen presentation, with their binding peptides largely originating from conserved regions of the viral genome, reducing the risk of variant escape.

[0033] Example 2 This embodiment discloses HLA-E-restricted SARS-CoV-2-specific CD8. + Application of T-cell epitope peptides in the preparation of SARS-CoV-2 epitope peptide vaccines.

[0034] Traditional vaccine development relies on CD8 molecules presented by classic HLA-I class molecules. + T-cell epitopes are highly polymorphic, but classic HLA-I molecules can only cover a limited population with a single epitope. This invention utilizes the low polymorphism of HLA-E molecules to significantly expand the immune response coverage of epitope peptides. Furthermore, viral epitopes presented by HLA-E molecules mostly originate from conserved regions, reducing the risk of immune escape due to viral mutations. This invention solves the problem of insufficient vaccine universality caused by the restrictive nature of classic HLA-I molecules, enabling the induction of specific CD8 in a broad population using a single epitope peptide. + T-cell responses provide a feasible basis for the development of broad-spectrum antiviral vaccines.

[0035] HLA-E restricted epitope peptides are specific amino acid sequences derived from the structural proteins of the SARS-CoV-2 virus. They can specifically bind to HLA-E molecules and induce CD8. + T-cell immune response. Specifically, this can be achieved using a synthetic peptide containing the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, which forms a complex with HLA-E molecules through in vitro assembly. Epitope peptide vaccines refer to vaccine formulations with synthetic peptides as the active ingredient, which activate specific CD8+ cells by directly presenting epitope peptides. + T cells do not rely on the intracellular processing of antigen-presenting cells.

[0036] Specifically, the method described in this embodiment involves screening for viral conserved epitope peptides with high affinity for HLA-E molecules, using them as core components of the vaccine. During vaccine preparation, the epitope peptides are obtained through chemical synthesis and combined with adjuvants or delivery systems to form the vaccine formulation. Once the vaccine enters the body, the epitope peptides can directly bind to HLA-E molecules on the surface of antigen-presenting cells, forming a stable complex, which is then absorbed by CD8+. + T-cell TCR recognition. This process bypasses the limitations of traditional vaccines that rely on classical HLA-I molecules to present antigens, and because HLA-E molecules have extremely low polymorphism in the population, it can cover a wider range of people. In addition, the selected epitope peptides are derived from conserved regions of the virus, which can maintain the ability to cross-recognize variant strains.

[0037] Traditional vaccines rely on classic HLA-I class molecules such as HLA-A or HLA-B to present antigens, but their high polymorphism limits the coverage of the population's immune response. For example, the HLA-A*02 restrictive epitope is only effective in specific populations, while the two major HLA-E alleles are nearly evenly distributed in the Han Chinese population, and their epitope binding characteristics are similar. Therefore, this embodiment overcomes the herd immunity differences caused by classic HLA restriction by targeting non-classical HLA-E molecules, while simultaneously utilizing conserved viral epitopes to enhance defense against variant strains. Existing inactivated or recombinant protein vaccines mainly activate humoral immunity, while this embodiment directly activates cellular immune responses through precisely designed epitope peptides, compensating for the shortcomings of traditional vaccines in inducing a long-lasting immune barrier.

[0038] Through the above technical solution, this invention solves the problem of low population coverage caused by HLA restriction in existing vaccines, achieving broader immune response coverage in a wider population through non-classical HLA-E molecules. Simultaneously, epitope peptides designed based on conserved viral regions can resist immune escape by variant strains, maintaining vaccine protective efficacy. This solution also directly activates CD8... + T cell responses established a cytotoxic killing mechanism against virus-infected cells, providing a new immune pathway for virus clearance.

[0039] Example 3 This embodiment presents a SARS-CoV-2 specific HLA-E / epitope peptide-complex tetramer, which is composed of four biotin-labeled HLA-E / epitope peptide-complex monomers and one avidin molecule. The complex monomer is formed by the in vitro assembly of an HLA-E molecular heavy chain with an activated biotin molecule linked to its C-terminus and β2 microglobulin. The amino acid sequence of the epitope peptide is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0040] Among them, the biotin-labeled HLA-E / epitope peptide-complex monomer refers to the introduction of activated biotin molecules into the C-terminus of the HLA-E heavy chain through chemical coupling, so that each monomer carries a biotin tag. This design can ensure that the complex monomer binds to avidin molecules in a directional manner to form a tetramer structure, while avoiding the biotin tag interfering with the conformation of the antigen binding groove.

[0041] In vitro assembly refers to the mixing and incubation of HLA-E heavy chain, β2 microglobulin and epitope peptide in a non-cellular environment. The correct folding of the complex monomers is achieved by pH regulation and temperature control. This process can precisely control the loading efficiency of epitope peptides and avoid the uncertainties caused by intracellular processing.

[0042] Among them, the binding of avidin molecules to four complex monomers refers to the use of the non-covalent binding property of high affinity between avidin and biotin to orient the four monomers to form a spatial multivalent structure. This structure can enhance the binding stability of the tetramer to TCR on the surface of T cells and improve detection sensitivity.

[0043] Specifically, after in vitro expression of the HLA-E heavy chain, an activated biotin molecule is linked to its C-terminus through site-directed modification. This biotin then incubates with β2-microglobulin and an epitope peptide in a specific buffer system, forming a complex monomer carrying the epitope peptide. The four monomers bind to a single avidin molecule via the biotin-avidin system, forming a tetrameric structure. This tetrameric structure binds to CD8 via a multivalent binding mode. + The interaction between T cell surface TCRs and their spatial conformation can mimic the natural antigen presentation process. Due to the low polymorphism of HLA-E molecules, the epitope peptides shown in SEQ ID NO: 1 or SEQ ID NO: 2 can cover individuals with different HLA-E alleles, achieving universal population-based detection.

[0044] Compared to existing technologies, traditional T-cell detection tools mainly rely on tetramers of classic HLA-I molecules. However, the detection of HLA-E-restricted T cells lacks specific tools because HLA-E, as a non-classical MHC I molecule, is more difficult to prepare as a tetramer. Traditional methods struggle to stably load specific peptides, resulting in insufficient detection specificity. Therefore, traditional methods cannot effectively solve the stability problem. This embodiment combines biotin-avidin directional coupling technology, providing an effective solution to the problem of poor stability of non-classical HLA tetramers.

[0045] Through the above technical solution, this application can specifically identify HLA-E-restricted CD8+ cells produced after SARS-CoV-2 infection. +During the detection process, the tetramer of T cells showed significantly better binding affinity to the TCR than the monomeric form, and was unaffected by individual HLA-E allele polymorphism. This tetramer can clearly distinguish epitope peptide-specific T cell populations in flow cytometry, providing a reliable tool for assessing vaccine efficacy and post-infection cellular immune response levels.

[0046] Preferably, the avidin molecule refers to a tetrameric protein capable of binding with biotin molecules with high affinity. Specifically, streptavidin or avidin can be used, which binds to four biotin-labeled HLA-E / epitope peptide-complex monomers through four binding sites. The fluorescein (PE) refers to a phycoerythrin-based fluorescent dye, specifically R-phycoerythrin or allophycocyanin conjugates. It covalently binds to the amino or thiol groups on the surface of the avidin molecule through chemical cross-linking, emitting a specific wavelength of fluorescence signal during flow cytometry detection.

[0047] Specifically, the tetrameric complex links four monomers into a spatially symmetrical structure via a fluorescein (PE)-labeled avidin molecule. When this complex interacts with CD8... + When TCRs bind to the surface of T cells, the fluorescence signal is captured by the photomultiplier tube of the flow cytometer and converted into an electrical signal. Since the maximum excitation wavelength of PE matches that of a 488nm laser, its emission spectrum is located near 575nm, compatible with the conventional detection channels of the flow cytometer, enabling the detection of low-frequency epitope peptides specific to CD8. + T cells can be effectively distinguished by fluorescence intensity thresholds. The high quantum yield of fluorescein PE allows a single tetrameric complex to carry multiple fluorescent molecules, thereby significantly improving the signal-to-noise ratio of the detection signal while maintaining the structural stability of the complex.

[0048] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments: This invention first applies the epitope prediction software NetMHCpan version 4.1 to analyze CD8 sequences in the SARS-CoV-2 whole genome sequence that may be HLA-E restricted. + T-cell 9-peptide epitopes were predicted, and the predicted SARS-CoV-2 9-peptides were screened using HLA-E*0103 and HLA-E*0101 transfection K562 cell lines to obtain high-affinity SARS-CoV-2 specific CD8+ cells containing either HLA-E*0103 or HLA-E*0101. + T-cell 9-peptide epitope.

[0049] Further ELISpot assays were used to identify HLA-E-restricted SARS-CoV-2 9-peptide epitopes. HLA-E / peptide-tetramer staining was used to detect the frequency of specific CTLs for HLA-E-restricted SARS-CoV-2 9-peptide epitopes in peripheral blood of COVID-19 recovered individuals, and the role of HLA-E-restricted epitope-specific CTL responses in COVID-19 was analyzed.

[0050] The present invention will now be described in detail, but this description is intended to explain rather than limit the invention.

[0051] 1. Software predicts HLA-E-restricted SARS-CoV-2 CD8 + T-cell 9-peptide epitope The complete genome sequence of SARS-CoV-2 can be obtained from the protein database on the NCBI website (Table 1). The online prediction software NetMHCpan Server version 4.1 (https: / / services.healthtech.dtu.dk / services / NetMHCpan-4.1 / ) was used to predict the HLA-E-restricted CD8+ of SARS-CoV-2. + Predicting T-cell epitopes.

[0052] NetMHCpan 4.1 Server is a deep neural network (DNN)-based tool for predicting MHC class I peptide binding, integrating binding affinity (BA) and mass spectrometry elution ligand (EL) data. Its training set contains 1,234,567 peptide-MHC complex data points, with the BA data covering 632,891 IC50 values ​​from 285 MHC alleles. 50 The EL data covers 601,676 mass spectrometry validation ligands for 412 MHC alleles. Core functions include: predicting peptide binding affinity (IC50). 50 This platform, capable of assessing ligand likelihood (with a percentage ranking ≤0.5% indicating strong binding) and generating 8-14 peptide predictions from whole-protein scans, has become a core platform for screening tumor neoantigen vaccines and infectious disease epitopes due to its high-precision prediction of non-classical MHC such as HLA-E and G, as well as cross-species MHC. Therefore, we selected HLA-E as the species / locus, HLA-E*0103 and HLA-E*0101 as the alleles, and a peptide length of 9 amino acids. The default strong binding threshold was set at the top 0.5% ranking, and the weak binding threshold at the top 2% ranking. Amino acid sequences of various SARS-CoV-2 proteins were then input for calculation.

[0053] The predicted SARS-CoV-2 9-peptide CD8, restricted by HLA-E in the Han Chinese population, was obtained. + T cell epitope peptides. Based on the software prediction results and the common anchoring amino acid pattern at positions 2 and 9 of HLA-E restricted 9-peptide epitopes, 27 9-peptides with high predicted affinity and 5 9-peptides reported in the literature were selected as candidate peptides (Table 2). Positive and negative control peptides were also selected based on literature reports (Table 3).

[0054] Table 1. Amino acid sequence information of different proteins of SARS-CoV-2

[0055] Table 2. Prediction of 9 HLA-E-restricted SARS-CoV-2 origin peptide epitopes.

[0056] Note: SB: Strongly binding peptides (top 0.5% ranked); WB: Weakly binding peptides (top 2.0% ranked). Table 3. Positive and negative controls for HLA-E-restricted SARS-CoV-2 9-peptide.

[0057] 2. Synthesis of the predicted 9 peptides derived from SARS-CoV-2. Based on software predictions, SARS-CoV-2 CD8 cells are restricted by HLA-E*0103 or HLA-E*0101. + Based on T-cell epitope results, Qiangyao Biotechnology Co., Ltd. synthesized 32 SARS-CoV-2-derived 9 peptides and 2 control 9 peptides. Each 9 peptide was synthesized at a dose of 5 mg, and the purity of all peptides was >90% as determined by RP-HPLC. The peptide powder can be stored long-term at -80℃. Before use, all 9 peptides were first dissolved in 20 μL of sterile dimethyl sulfoxide (DMSO), and after complete dissolution, sterile PBS was added to prepare a solution with a final concentration of 2 mM. After aliquoting, the solution can be frozen at -80℃ for later use.

[0058] 3. Screening for high-affinity SARS-CoV-2 CD8 cells using HLA-E / K562 stable cell binding assays. + T-cell 9 peptide 3.1 Lentiviral transfection to construct stable K562 cells with HLA-E*0103 or HLA-E*0101 K562 cells are a human chronic myeloid leukemia cell line, characterized by the absence of HLA-I antigen molecules. Therefore, they are widely used in studies of the in vitro expression and function of HLA-E molecules. Lentiviral transfection was used to transform HLA-E*0103 and HLA-E*0101 into K562 cells, respectively, to obtain stable expression of either HLA-E*0103 or HLA-E*0101 on the cell surface, which was then used to screen for high-affinity SARS-CoV-2 9-peptides of HLA-E*0103 or HLA-E*0101. First, overexpressing lentiviral clones were prepared, and Real-time PCR was used to detect the expression abundance of the target gene HLA-E*0103 or HLA-E*0101 mRNA in K562 cells. The upstream primer sequence for the target gene was GAGAGGAGCAGAGATACAC, and the downstream primer sequence was TCCAGAGACCACAGATCC. Quantitative PCR results showed that the expression abundance of HLA-E*0103 or HLA-E*0101 genes in K562 cells was higher than that in the control group, suggesting that lentiviral transfection of HLA-E*0103 or HLA-E*0101 resulted in stable expression on the surface of K562 cells. Based on the results of the lentiviral infection pre-experiment, GFP expression was observed under a fluorescence microscope 72 hours after infection. Subsequently, appropriate concentrations of puromycin were used for selection for at least 48 hours. When cell fluorescence efficiency reached 100% or no further cell death occurred, the puromycin concentration was at least halved, and cell culture was continued to expand. Cells with normal results were cryopreserved for propagation after qPCR identification, thus completing the construction of stable mixed clones.

[0059] The expression levels of HLA-E*0103 or HLA-E*0101 molecules on the surface of transfected K562 cells were detected using FCM, as shown below: 2×10 5 K562 cells, K562 cells transfected with HLA-E*0103 or HLA-E*0101, were incubated with PE-labeled mouse anti-human HLA-E mAb (3D12), along with isotype control tubes. The cells were incubated at 4°C for 30 min, washed with FCM wash buffer, centrifuged, and then vortexed before adding 300 μl of FCM fixative. Flow cytometry analysis was performed. Results showed that the control group K562 cells showed almost no expression of HLA-E*0103 or HLA-E*0101 molecules, while the expression of HLA-E*0103 or HLA-E*0101 molecules was significantly increased in the HLA-E overexpression lentivirus group. The constructed HLA-E stable transfected cells can be used as antigen-presenting cells (APCs), providing a powerful tool for further screening of HLA-E-restricted SARS-CoV-2 9-peptide epitopes.

[0060] 3.2 HLA-E / K562 stable cell binding assays yielded two high-affinity SARS-CoV-2-derived 9-peptides, either HLA-E*0103 or HLA-E*0101. The binding of exogenous peptides to HLA-E molecules on the surface of HLA-E / K562 stable transgenic cells increases HLA-E expression; the stronger the binding, the higher the HLA-E expression. Therefore, the increased HLA-E expression level on HLA-E / K562 stable transgenic cells directly reflects the affinity of the antigen peptide for HLA-E*0103 or HLA-E*0101 molecules. Specifically: After resuscitation, the HLA-E / K562 stable cell line was cultured to the logarithmic growth phase, then transferred to 26°C and 5% CO2 for overnight culture to induce HLA-E expression. The cell density was adjusted to 5 × 10⁶ cells / year using serum-free RPMI 1640 medium. 6 Add 0.1 ml of the solution to each well of a 96-well cell culture plate, approximately 5 × 10⁶ cells / ml. 5 Cells were added to each well; a synthetic SARS-CoV-2 candidate 9-peptide (final concentration 100 μM) and β2m (final concentration 2.5 μg / ml) were added simultaneously; a blank control was set up, i.e., no 9-peptide was added, only cells and β2m were added; a positive control was set up, i.e., a known HLA-E-restricted HLA-A2 leader sequence 9-peptide was added (Table 3); a negative control was set up, i.e., a non-HLA-E-restricted MAGE-1 derived 9-peptide reported in the literature was added (Table 3); cells were cultured at 26℃ and 5% CO2 for 18 h; after centrifugation and washing, mouse anti-human HLA-E-APC-mAb (3D12) was added, 5 μl / tube, and incubated at 4℃ for 30 min; after washing, flow cytometry was used for detection; the FI value was calculated, and a FI value > 1 was used to determine a high affinity epitope, the calculation formula is as follows: FI = (Average fluorescence intensity of APC in experimental wells - Average fluorescence intensity of APC in blank control wells) / Average fluorescence intensity of APC in blank control wells The results showed that the positive control peptide had a binding affinity (FI) greater than 3 with HLA-E / K562 cells, while the negative control peptide had a FI value less than 1. Among the 32 candidate SARS-CoV-2 9 peptides tested, VMVELVAEL (derived from NSP1) and SMQNCVLKL (derived from NSP5) had FI values ​​greater than 1 with HLA-E / K562 cells. Therefore, these two SARS-CoV-2-derived 9 peptides can be identified as high-affinity HLA-E binding peptides. Figure 1 (Table 4).

[0061] Figure 1 The assay showed HLA-E / K562 stable cell binding to screen HLA-E high-affinity SARS-CoV-2 CD8 cells.+ The flow cytometry results of the 9-peptide T-cell assay are shown in the bar chart. In each chart, the five bars from left to right represent the fluorescence intensity of HLA-E / K562 stable transgenic cells expressing HLA-E*0103 or HLA-E*0101 molecules after loading the experimental peptide, the isotype control, the negative control peptide MAGE-1 (EADPTGHSY), and the positive control peptide HLA-A2 leader sequence 9-peptide (VMAPRTLVL). Therefore, the greater the rightward shift of the experimental peptide's peak compared to the isotype and negative control peaks, the higher the affinity of that peptide for HLA-E*0103 or HLA-E*0101, and the more likely it is to be an HLA-E-restricted SARS-CoV-2 CD8+. + T-cell 9-peptide epitope.

[0062] Table 4. HLA-E / K562 cell binding assay for screening HLA-E*0103 molecule with high affinity for SARS-CoV-2 9-peptide.

[0063] *FI: Fluorescence index, FI = (Average PE fluorescence index of loaded specific peptides - Average PE fluorescence index of unloaded peptides) / (Average PE fluorescence index of unloaded peptides). FI ≥ 1 indicates a high-affinity epitope peptide. SB: Strongly binding peptides (top 0.5% ranked); WB: Weakly binding peptides (top 2.0% ranked).

[0064] 4. Enzyme-linked immunospot assay (ELISpot) for identifying HLA-E-restricted SARS-CoV-2 CD8. + T-cell 9-peptide epitope 4.1 Isolation of peripheral blood mononuclear cells (PBMCs) from peripheral blood of individuals recovering from COVID-19 According to the Chinese clinical diagnostic criteria for COVID-19: 1) Positive SARS-CoV-2 virus nucleic acid or antigen test; or positive SARS-CoV-2 virus isolation and culture; or SARS-CoV-2 virus-specific IgG antibody level in the convalescent phase is 4 times or more higher than that in the acute phase; 2) SARS-CoV-2 virus infection-related clinical manifestations. Information on the population was collected based on the COVID-19 convalescent population according to different clinical subtypes and vaccination status. 8 mL of peripheral blood was aseptically collected in EDTA anticoagulant tubes and diluted with an equal volume of physiological saline. Add 4 ml of lymphocyte separation medium (Tianjin Haoyang Biotechnology Co., Ltd.) to a 15 ml centrifuge tube beforehand. Use a bent pipette to slowly add anticoagulated blood onto the lymphocyte separation medium. Centrifuge at 2000 rpm for 20 min without brake. Carefully aspirate the white cloud layer at the interface between the two layers. Add physiological saline to 50 ml to wash the cloud layer cells. Centrifuge at 2000 rpm for 10 min to obtain PBMCs from COVID-19 convalescent individuals. The cells can be used directly for subsequent experiments or resuspended in cryopreservation solution (90% FCS, 10% DMSO) and stored in liquid nitrogen for later use.

[0065] 4.2 Identification of HLA-E-restricted SARS-CoV-2 CD8+ nucleotides by IFN-γ ELISApot that can stimulate CTL responses in COVID-19 convalescent individuals + T-cell 9-peptide epitope 4.2.1 CD8+ cells in PBMCs from COVID-19 convalescent individuals were sorted using an immunomagnetic bead (Dynal) negative sorting method. + T cells, specifically, are prepared by taking 1×10⁶ cells. 7 After resuspending PBMCs from COVID-19 convalescent patients, 20 μL of FCS and 20 μL of antibody mixture were added, and the mixture was incubated at 4°C for 20 min. The cells were washed with 5 mL of buffer, centrifuged, and then resuspended in 1 mL of buffer. This resuspended cells were added to pre-washed Dynabeads, mixed, and incubated at room temperature for 15 min, inverting the mixture every 5 min. The cells were then incubated on a magnetic rack for 2 min, and the supernatant was collected. The cells were resuspended again with 1 mL of buffer, incubated on a magnetic rack for 2 min, and the supernatant was collected again. This process was repeated three times. After centrifugation, the cells were resuspended in 10% FCS RPMI 1640 medium, resulting in CD8 cells without magnetic beads or antibodies on the cell surface. + T cells.

[0066] 4.2.2 Loading HLA-E / K562 stable transgenic cells with high affinity for SARS-CoV-2 candidate 9 peptides. The specific loading method was as follows: HLA-E / K562 stable transgenic cells were cultured in 10% FCS RPMI 1640 medium at 26°C overnight. The corresponding high affinity SARS-CoV-2 candidate 9 peptides (final concentration 100 μM) were added, and the cells were cultured in Opti-MEM medium at 26°C for 16 h. After washing with sterile PBS to remove excess peptide fragments, the peptides were counted.

[0067] 4.2.3 IFN-γ ELISpot (Dakow Biotech) induces CD8 in CD8 receptor antagonism against a high-affinity 9-peptide candidate SARS-CoV-2. + The ability of T cells to secrete IFN-γ was measured, and IFN-γ was used as the detection target to detect the HLA-E-restricted SARS-CoV-2 9-peptide against CD8 in COVID-19 convalescent patients. + The specific stimulation of T cells involves adding magnetic beads to sort CD8 cells. + T cells (5×10) 4 (5 × 10⁶ cells / well), along with HLA-E / K562 stable cells preloaded with SARS-CoV-2 candidate 9 peptides (5 × 10⁶ cells / well). 4 / well), co-cultured at 37℃ and 5% CO2 for 36h; positive and negative controls were set up simultaneously, with PHA (final concentration 10ug / ml) added to the positive control wells and CD8 added only to the negative control wells. + T cells were cultured, and the liquid was removed and pre-cooled deionized water was added. Cells were lysed in a hypotonic environment at 4°C for 10 min. After washing, biotinylated anti-human IFN-γ antibody (100 μl / well) was added, and the mixture was incubated at 37°C for 1 h. After washing, stretchavidin-HRP (100 μl / well) was added, and the mixture was incubated at 37°C for 1 h. After washing, freshly prepared AEC chromogenic solution (100 μl / well) was added, and the mixture was incubated at room temperature in the dark for 30 min. The chromogenic process was stopped by rinsing with tap water, and the cells were air-dried. Spots in each well were counted using an ELISpot reader (CTL Corporation, USA). According to literature, a positive response was defined as ≥50 SFCs / 1010 per well. 6 If effector cells are present and the number of spots is at least three times that of the negative control wells, then the positive response candidate peptide can be identified as HLA-E-restricted CD8. + T cell-specific SARS-CoV-2 9-peptide epitopes. Normalized to SFCs / 10 using the following formula. 6 cell: SFC / 10 6Cells = [(Number of spots in positive control wells - Number of spots in negative control wells) / Total number of effector cells added to each well] × 10 6 The results showed that all nine HLA-E high-affinity SARS-CoV-2 candidate peptides could stimulate CTLs in COVID-19 convalescent patients to secrete IFN-γ to varying degrees. The frequencies (mean ± standard deviation) of HLA-E-restricted SARS-CoV-2 epitope-specific CTLs in PBMCs of COVID-19 convalescent patients were 73 ± 1168 SFC / 10 6 Cells (SEQ ID NO: 1 VMVELVAEL), 193±2331 SFC / 10 6 Cells (SEQ ID NO: 2 SMQNCVLKL) Figure 2 ). Figure 2 This image shows the frequency of IFN-γ secretion by peripheral blood CTLs in COVID-19 convalescent individuals stimulated by an HLA-E high-affinity SARS-CoV-2-derived 9-peptide detected by ELISpot. Taking the results from COVID-19 convalescent patient No. 16 as an example, each well in the image, from left to right, represents the SEQ ID NO: 1 (VMVELVAEL) stimulation well, the SEQ ID NO: 2 (SMQNCVLKL) stimulation well, the PHA positive control well, and the unstimulated blank control well. A higher number of spots per well indicates a stronger CTL response induced by this SARS-CoV-2 epitope in COVID-19 convalescent individuals. Therefore, both 9-peptides were identified as HLA-E-restricted SARS-CoV-2 CD8+. + T-cell epitopes.

[0068] 4.2.4 A comparison was made between the frequency of IFN-γ secretion by HLA-E-restricted epitope-specific CTLs in peripheral blood of uninfected normal controls (PBMCs specimens frozen in our laboratory before 2020 that were not infected with SARS-CoV-2) and individuals recovering from COVID-19. The results showed that the frequency of IFN-γ secretion by HLA-E-restricted epitope-specific CTLs was significantly higher in the recovery population than in the uninfected healthy population. Figure 3 ). Figure 3The chart shows a statistical comparison of the frequency of IFN-γ secretion by HLA-E-restricted SARS-CoV-2 9-peptide epitope-specific CTLs in peripheral blood between COVID-19 convalescents and uninfected healthy individuals. The chart compares the frequency of IFN-γ secretion by two HLA-E-restricted SARS-CoV-2 9-peptide epitope-specific CTLs in peripheral blood between COVID-19 convalescents and uninfected healthy individuals (x-axis) and (y-axis). The results show that the frequency of IFN-γ secretion by both 9-peptide epitope-specific CTLs was significantly higher in the COVID-19 convalescent population than in the uninfected healthy population, suggesting that the COVID-19 convalescent population has a higher level of CD8 specific antigen against SARS-CoV-2. + T-cell immune memory, and CD8 in recovered patients + The T-cell immune barrier has been basically established. 5. HLA-E*0103 / epitope tetramer staining to detect the frequency of SARS-CoV-2 epitope-specific CTLs in peripheral blood of COVID-19 convalescent patients. 5.1 Based on the epitope identification results, HLA-E / epitope peptide tetramers were constructed for two SARS-CoV-2 9-peptide epitopes (Jiangsu Nantong Biaoyuan Biotechnology Co., Ltd.). Through monomer identification and biotin-avidin tetramer synthesis, the binding rate of biotinylated MHC monomers to streptavidin was determined to be ≥80%. Finally, the tetramers were labeled with PE.

[0069] 5.2 The frequency of HLA-E-restricted SARS-CoV-2 9-peptide epitope-specific CTLs in peripheral blood of COVID-19 convalescent individuals was detected using the HLA-E / epitope tetramer staining method, as follows: PBMCs (1×10⁻⁶) from COVID-19 convalescent individuals or healthy controls were collected. 6 After washing, centrifuge at 1200 rpm for 5 min, add HLA-E / epitope peptide tetramer-PE (2 μl / tube), and incubate at room temperature in the dark for 15 min. After washing, add anti-human CD8-APC and CD3-PerCP-Cy5.5 antibodies respectively, and set up isotype control tubes. Incubate at 4℃ in the dark for 30 min. After washing, add 100 μl of fixative and mix well. Analyze by flow cytometry.

[0070] Tetramer staining results showed that both SARS-CoV-2 9-peptide epitope-specific CTLs were detectable in PBMCs of individuals recovering from COVID-19. Figure 4 ). Figure 4This image shows the frequency of HLA-E-restricted SARS-CoV-2 9-peptide epitope-specific CTLs in the peripheral blood of individuals recovering from COVID-19, using tetramer staining as an example. NovoExpress 1.4.1 software was used to gate the PBMCs cell population on the SSC and FSC scatter plots, and then gated the CD3+ cells. - CD56 - Cell population, and finally gating analysis of CD8. + (x-axis) Tetramer + The cell population proportion (vertical axis) represents the frequency of HLA-E-restricted SARS-CoV-2 9-peptide epitope-specific CTLs in the peripheral blood of individuals recovering from COVID-19. A higher frequency indicates a stronger CTL response induced by this SARS-CoV-2 9-peptide epitope in recovered COVID-19 individuals. The frequencies (mean ± standard deviation) of HLA-E-restricted SARS-CoV-2 epitope-specific CTLs in PBMCs of recovered COVID-19 individuals were 1.68 ± 1.52 (SEQ ID NO: 1 VMVELVAEL) and 1.24 ± 1.52 (SEQ ID NO: 2 SMQNCVLKL), respectively.

[0071] 5.3 The response frequency of HLA-E-restricted SARS-CoV-2 epitope-specific CTLs in PBMCs of COVID-19 convalescent individuals can be used to evaluate the body's immune memory and immune response capacity against SARS-CoV-2. A higher CTL frequency indicates a stronger immune barrier established in the convalescent population. Furthermore, the two HLA-E-restricted SARS-CoV-2-specific epitopes are effective immunodominant epitopes in the population and can serve as potential candidate antigens for peptide vaccines. Figure 5 ).

[0072] In this figure, A represents a statistical comparison of the frequencies of HLA-E-restricted SARS-CoV-2 9-peptide epitope-specific CTLs in peripheral blood of COVID-19 convalescent patients and uninfected healthy controls (x-axis) (y-axis). The results showed that the frequencies of SARS-CoV-2 epitope-specific CTLs in the convalescent population were all higher than those in the uninfected healthy controls. Specifically, the frequency of the SEQ ID NO: 2 SMQNCVLKL epitope-specific CTL was significantly higher in the convalescent population than in the uninfected healthy controls. P<0.05). B represents a statistical comparison of the frequency of HLA-E-restricted SARS-CoV-2 epitope-specific CTLs in peripheral blood of COVID-19 convalescents with different disease levels at infection. The frequencies of epitope-specific CTLs in peripheral blood were compared between individuals diagnosed with mild (Mild), severe (Severe), and uninfected normal controls (x-axis) at infection (y-axis). The results showed that the frequency of epitope-specific CTLs was higher in the mild group than in the severe group, and the frequency of the SEQ ID NO: 1 VMVELVAEL epitope-specific CTL in the mild group was significantly higher than that in the severe group and the uninfected normal control group (…). P <0.05). C represents a statistical comparison of the frequency of HLA-E-restricted SARS-CoV-2 epitope-specific CTLs in peripheral blood of symptomatic and asymptomatic COVID-19 convalescents. The frequencies of epitope-specific CTLs in peripheral blood were compared between the symptomatic group, asymptomatic group, and uninfected normal controls (x-axis). The results showed that the frequency of epitope-specific CTLs was higher in the symptomatic group than in the asymptomatic group. D represents a statistical comparison of the frequency of HLA-E-restricted SARS-CoV-2 epitope-specific CTLs in peripheral blood of COVID-19 convalescents who received 2 or 3 doses of SARS-CoV-2 vaccine. The frequencies of epitope-specific CTLs in peripheral blood were compared between the 2-dose group, the 3-dose group, and uninfected normal controls (x-axis). The results showed that the frequency of epitope-specific CTLs was higher in the 3-dose group than in the 2-dose group or the uninfected group.

[0073] In summary, these results suggest that the identified HLA-E-restricted SARS-CoV-2 9-peptide epitope-specific CTL response plays an immunoprotective role in SARS-CoV-2 infection. An immune protective barrier based on the HLA-E-restricted SARS-CoV-2 epitope-specific CTL response has been widely established in the convalescent population, and three doses of SARS-CoV-2 vaccination can provide a longer-lasting and stronger cellular immune protection effect. Because HLA-E molecular polymorphism is very limited and it is a ubiquitous HLA allele locus in the population, the identification of the HLA-E-restricted SARS-CoV-2 CD8... + T-cell epitopes not only have important theoretical significance in revealing the pathogenesis of COVID-19, but also provide important reference value for assessing the level of immune barrier establishment in the COVID-19 convalescent population, provide reliable experimental basis for designing novel SARS-CoV-2 peptide vaccines that are more specific and effective for the Chinese population, and provide important theoretical basis for formulating new clinical treatment strategies. They also have good prospects for development and application in the field of COVID-19 specific immunotherapy.

[0074] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A class of HLA-E-restricted SARS-CoV-2-specific CD8 receptors + T-cell epitope peptide, characterized in that... These epitope peptides, after binding to and being presented with HLA-E molecules, can induce CD8. + T cells generate cellular immune responses.

2. The HLA-E-restricted SARS-CoV-2-specific CD8 receptor agonist according to claim 1 + T-cell epitope peptide, characterized in that... This type of epitope peptide binds complementaryly to the antigen-binding groove formed by the α1 and α2 domains of the HLA-E molecule. During the binding process, the hydrophobic residues of the 2nd and 9th amino acids of the epitope peptide are embedded in the anchoring sites of the HLA-E molecule binding groove, while the polar residues in the middle region form a hydrogen bond network with the HLA-E molecule, forming a stable complex structure.

3. The HLA-E-restricted SARS-CoV-2-specific CD8 receptor agonist according to claim 1 + T-cell epitope peptide, characterized in that... The amino acid sequence of the epitope peptide is shown in SEQ ID NO:1 or SEQ ID NO:

2.

4. The HLA-E-restricted SARS-CoV-2-specific CD8 receptor agonist according to any one of claims 1-3 + Application of T-cell epitope peptides in the preparation of SARS-CoV-2 epitope peptide vaccines.

5. The application according to claim 4, characterized in that, The SARS-CoV-2 epitope peptide vaccine described above is capable of inducing the production of epitope peptide-specific CD8. + T-cell epitope peptide vaccine.

6. A monomer of an HLA-E / epitope peptide complex, characterized in that, The HLA-E-restricted SARS-CoV-2-specific CD8 receptor agonist described in any one of claims 1-3 + The complex is composed of T-cell epitope peptides and a complex monomer; the complex monomer is a structure assembled in vitro from an HLA-E molecular heavy chain with an activated biotin molecule linked to its C-terminus and β2 microglobulin.

7. A SARS-CoV-2 specific HLA-E / epitope peptide-complex tetramer, characterized in that, It consists of one avidin molecule and four biotin-labeled HLA-E / epitope peptide complex monomers; The HLA-E / epitope peptide complex monomer is the HLA-E / epitope peptide complex monomer according to claim 6.

8. The SARS-CoV-2-specific HLA-E / epitope peptide-complex tetramer according to claim 7, characterized in that, The avidin molecule is labeled with fluorescein PE.

9. The SARS-CoV-2-specific HLA-E / epitope peptide-complex tetramer according to claim 7, characterized in that, This HLA-E / epitope peptide-complex tetramer can bind to epitope-specific CD8. + TCRs bind to the surface of T cells.

10. The SARS-CoV-2 specific HLA-E / epitope peptide-complex tetramer according to any one of claims 7-9 in the detection of SARS-CoV-2 epitope peptide-specific CD8 + Applications in T cell frequency.

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