A monkeypox virus antigen epitope peptide and application thereof

Abstract

The present application belongs to the technical field of immunotherapy, and particularly relates to a monkeypox virus antigen epitope peptide and application thereof. The present application aims to solve the technical problem that no T cell antigen epitope peptide for a monkeypox virus universal vaccine has been developed in the field of monkeypox virus. The technical scheme of the present application is a monkeypox virus antigen epitope peptide, the amino acid sequence of which is shown in SEQ ID No. 2. The antigen epitope peptide provided by the present application has strong immunogenicity and can induce antigen-specific CD8+ T cells; the antigen epitope peptide can be directly loaded into antigen presenting cells, can activate T cells, effectively induce T cell immunity, and can be used for research and development, preparation and drug research and development, and clinical treatment of a monkeypox virus universal vaccine.

Description

Technical Field

[0001] This invention belongs to the field of immunotherapy technology, specifically relating to a monkeypox virus cell antigen epitope peptide and its application. Background Technology

[0002] Monkeypox is a zoonotic viral disease caused by the monkeypox virus (MPXV), which belongs to the orthopoxvirus family of the poxviridae family. MPXV was first discovered in 1958 in research monkeys shipped from Singapore, which may explain the name "monkeypox." However, the natural hosts of MPXV are more likely rodents and other small mammals. The orthopoxvirus genus also includes smallpox virus (VARV), and human monkeypox presents with symptoms similar to smallpox, but with a lower mortality rate. Sporadic cases of human MPXV were first discovered in a few African countries in the 1970s, but the virus has spread widely across the continent over the past 20 years. Since May 2022, several countries have reported confirmed cases of monkeypox. China reported its first cases on June 25, September 6, and September 16, 2022, all of which were imported cases. Simultaneously, the number of MPXV cases worldwide has increased dramatically, leading the World Health Organization (WHO) to declare the monkeypox outbreak a global health emergency. As of June 2025, more than 100 countries and regions worldwide had reported 127,905 confirmed cases and 283 deaths, making it one of the most serious pandemics of our time.

[0003] Human monkeypox virus is a zoonotic orthopox virus that presents with clinical manifestations similar to smallpox. The incubation period for monkeypox is 5-21 days. Infected individuals typically begin with flu-like symptoms such as fever, headache, fatigue, muscle aches, and swollen lymph nodes. Swollen, soft, and relatively fixed lymph nodes (1-4 cm in diameter) appear in the submandibular, cervical, or groin areas. Monkeypox is often misdiagnosed as chickenpox or smallpox (up to 50% of cases in the Democratic Republic of Congo are misdiagnosed). The main difference between human monkeypox and smallpox is that human monkeypox virus causes swollen lymph nodes, often accompanied by fever; varicella-zoster virus lesions are usually superficial, with irregular borders, and can appear simultaneously in multiple stages on any part of the body, rapidly evolving from macules to scabs within 24 hours. The central genomic region of orthopox virus mainly consists of highly conserved essential genes. The central genomic region of monkeypox virus DNA is 101,466 bp in length and shares 96.3% homology with the corresponding part of the smallpox virus genome.

[0004] Compared to antibody and CD4+ T cell immune responses, research on the CD8+ T cell immune response to monkeypox virus is relatively limited. Early studies mainly focused on detecting activation markers of CD8+ T cells and using ELISPOT (Enzyme-Linked ImmunoSpot Assay), while little is known about the antigenic epitopes of CD8+ T cells. The acquired immune response plays a crucial role in combating monkeypox virus infection, including B cell antibody production, the helper and effector roles of CD4+ T cells (helper T cells), and the cytotoxic effect of CD8+ T cells (cytotoxic T cells) in killing and clearing virus-infected cells. In rhesus monkeys, smallpox virus-specific B cell responses help protect against fatal monkeypox virus infection. Epidemiological studies have further demonstrated that smallpox vaccines can protect against other poxvirus infections, including monkeypox virus. Following vaccination, smallpox virus-specific memory lymphocyte and antibody levels can persist for more than 50 years. However, more than 20 years after vaccination, only about 50% of vaccinated individuals had neutralizing antibody titers greater than 1:32, indicating that protective immunity against smallpox may weaken over time, and cross-protective immunity against monkeypox may also weaken. CD4+ T cells, especially T follicular helper cells (Tfh), play an important role in enhancing the differentiation of memory B cells into antibody-secreting cells. After vaccinia vaccination, memory CD4+ T cells can survive for 50 years or even longer, with an estimated half-life of 8-15 years. These vaccinia virus-specific CD4+ T cells can produce interferon-gamma (IFN-γ) and tumor necrosis factor (TNF) upon stimulation. However, there is no direct correlation between the number of virus-specific CD4+ T cells and the titer of anti-vaccinia virus antibodies. In rhesus monkeys infected with simian immunodeficiency virus, if the CD4+ T cell count is less than 300 cells / mm², the antibody titer is significantly lower. 3 In this case, the vaccinia virus cannot produce vaccinia virus-specific immunoglobulin G, and the host may die upon challenge with monkeypox virus. In contrast, a patient recently infected with monkeypox virus who received antiretroviral therapy with human immunodeficiency virus type 1 had a CD4+ T cell count higher than 700 cells / mm². 3 Furthermore, no severe disease outcomes were observed. These results indicate that, in addition to antibodies, T cells play a crucial role in regulating monkeypox virus severity. However, further research is needed to fully understand the function of T cells in the monkeypox virus infection process.

[0005] Therefore, research on monkeypox virus CD8+ T cell antigenic epitope peptides and their applications has significant scientific and clinical value. CD8+ T cells recognize viral antigenic peptides (pMHC) presented by major histocompatibility complex class I molecules through their T cell receptors, thereby activating and killing virus-infected cells, playing a crucial role in antiviral immunity. Although current vaccinia virus-based vaccines offer some cross-protection against monkeypox virus infection, this protective effect weakens over time, and its efficacy varies among individuals with different immune states. Of particular note is that monkeypox virus, as a large DNA virus, encodes numerous proteins that may be involved in immune evasion, making antibody-mediated humoral immunity alone insufficient to provide complete protection. Therefore, identifying conserved antigenic epitope peptides that can effectively activate CD8+ T cells is crucial for developing novel monkeypox vaccines and immunotherapy strategies. The identification of these epitope peptides not only helps design universal vaccines that induce durable T-cell immunity, overcoming the challenges posed by potential viral mutations, but also provides additional protective measures for immunocompromised individuals. Furthermore, T-cell immunotherapies developed based on these epitope peptides, such as peptide vaccines or adoptive T-cell therapy, hold promise as a complement to traditional antibody therapy, providing crucial protection, especially in cases of insufficient antibody response. A deeper understanding of the characteristics and regulatory mechanisms of monkeypox virus-specific CD8+ T-cell responses will also provide important evidence for optimizing existing vaccines, evaluating the effectiveness of immune protection, and ultimately promoting the establishment of more effective monkeypox control strategies. Summary of the Invention

[0006] The technical problem to be solved by this invention is that there is currently no T-cell antigen epitope peptide developed for a universal vaccine against monkeypox virus.

[0007] The technical solution of the present invention is a monkeypox virus antigen epitope peptide, the amino acid sequence of which is shown in SEQ ID No. 2.

[0008] Furthermore, the present invention also provides a nucleic acid molecule encoding the said antigenic epitope peptide.

[0009] The present invention also provides a pMHC complex containing the said antigenic epitope peptide.

[0010] Furthermore, the pMHC complex is obtained by refolding an MHC monomer and the antigenic epitope peptide.

[0011] In this mixture, MHC monomers and antigenic epitope peptides are mixed in equal volumes.

[0012] Furthermore, the concentration of the MHC monomer before mixing was 200 μg / mL, and the concentration of the antigenic epitope peptide before mixing was 400 μM.

[0013] The present invention further provides an antigen epitope peptide-antigen presenting cell complex, which is an antigen presenting cell with the antigen epitope peptide loaded on its surface.

[0014] The antigen-presenting cells are CD8+ T cells.

[0015] Preferably, the CD8+ T cells are T2-A2 cells.

[0016] The antigen-presenting cells are CD8+ T cells.

[0017] Preferably, the CD8+ T cells are T2-A2 cells.

[0018] The present invention also provides the use of the above-mentioned antigenic epitope peptide, the nucleic acid molecule encoding the antigenic epitope peptide, the pMHC complex and / or the antigenic peptide-antigen presenting cell complex in the preparation of monkeypox virus drugs.

[0019] The present invention also provides the application of the above-mentioned antigenic epitope peptide, the nucleic acid molecule encoding the antigenic epitope peptide, the pMHC complex and / or the antigenic peptide-antigen presenting cell complex in screening monkeypox virus drugs.

[0020] The present invention further provides the application of the above-mentioned antigenic epitope peptide, the nucleic acid molecule encoding the antigenic epitope peptide, the pMHC complex and / or the antigenic peptide-antigen presenting cell complex in the preparation of monkeypox virus vaccines.

[0021] The present invention further provides the application of the above-mentioned antigenic epitope peptide, the nucleic acid molecule encoding the antigenic epitope peptide, the pMHC complex and / or the antigenic peptide-antigen presenting cell complex in the preparation of a medicament for evaluating the efficacy of monkeypox virus vaccination.

[0022] The beneficial effects of this invention are as follows: This invention provides a monkeypox virus T-cell antigenic epitope peptide, which has strong immunogenicity and can induce antigen-specific CD8+ T cells; it can assemble with HLA-A2 heavy chain and HLA-A2 light chain β2m protein to form a pMHC complex; or it can be directly loaded onto antigen-presenting cells, which can activate T cells, effectively induce T-cell immunity, avoid immune escape of monkeypox virus mutant strains, and specific CD8+ T cells can be detected in monkeypox virus convalescent patients. It can be used for the development, preparation, drug development, and clinical treatment of universal monkeypox virus vaccines.

[0023] This invention also utilizes the monkeypox virus CD8+ T cell antigenic epitope peptide to prepare a pMHC complex with a PE fluorescent channel. This complex can be used to detect antigen-specific T cells in the peripheral blood of monkeypox virus vaccine recipients and recovered patients, and for in vitro T cell activation experiments. These monkeypox virus CD8+ T cell antigenic epitope peptides can be used to prepare universal vaccines against various monkeypox virus mutant strains, monkeypox virus-related immunoassays, and broad-spectrum therapeutic drugs. These monkeypox virus CD8+ T cell antigenic epitope peptides, prepared as a pMHC complex with a PE fluorescent channel, or directly loaded onto antigen-presenting cells to activate T cells, can be used for monkeypox virus vaccine development, preparation, drug development, and clinical treatment. Applications include:

[0024] 1) Development and preparation of monkeypox virus vaccines: After mutation, these multiple T-cell epitopes can induce the body to generate an immune response and produce antigen-specific T cells. Therefore, these T-cell epitopes are candidate antigenic epitope peptides for universal monkeypox virus vaccines.

[0025] 2) Detection of cellular immune function against monkeypox virus infection: The detection of monkeypox virus antigen-specific T cells in the subject indicates that the body has developed T cell immune function. The proportion of antigen-specific CD8+ T cells labeled with a fluorescent channel pMHC complex prepared from the antigen epitope peptide can be used to evaluate the strength of the body's T cell immune function and the likelihood of monkeypox virus infection.

[0026] 3) Assess the effectiveness of vaccination: The detection of monkeypox virus antigen-specific T cells in the recipient indicates that the body has developed T cell immune function. Based on the proportion of these cells, the possibility of reinfection with monkeypox virus can be assessed.

[0027] 4) Monitoring disease condition: It can be used to monitor changes in the condition of close contacts, medical observers, and suspected and confirmed patients.

[0028] 5) Prognosis: If the body cannot produce a T-cell immune response, or if the proportion of antigen-specific T cells continues to decrease, the prognosis is poor. Attached Figure Description

[0029] Figure 1 A: Statistical graph of T2-A2 antigen presentation of two monkeypox virus epitope peptides; B: Statistical graph of detection of pMHC complex formation by two monkeypox virus T cell epitope peptides. Blank control: no peptide added; Negative control: EB virus, SEQ ID No. 3, IVTDFSVIK; Positive control: Influenza A M1 peptide: SEQ ID No. 4, GILGFVFTL (the same below).

[0030] Figure 2Immunogenicity identification of monkeypox virus T-cell antigenic epitopes. A: CD69 expression level after 16 hours; B: CD137 expression level after 16 hours.

[0031] Figure 3 A: Antigen-specific T cell generation was detected after stimulation with antigen epitope peptides; B: Antigen-specific T cell generation can be detected by using a fluorescent channel tetramer after 7 days of stimulation of CD8+ T cells in healthy individuals with antigen epitopes.

[0032] Figure 4 The percentage of T2 cell apoptosis mediated by epitope stimulation. A: Percentage of T2A2 cells undergoing apoptosis after 7 days of stimulation and culture; B is a statistical graph of A.

[0033] Figure 5 1. To assess the proportion of specific CD8+ T cells in smallpox vaccine recipients 20 years later; A: Flow cytometry detection of specific CD8+ T cells of the above two types (antigen-specific CD8+ T epitopes that can be produced when T cells are activated) peptides in HLA-A2+ smallpox vaccine recipients; B is a statistical graph of A. Detailed Implementation

[0034] T2-A2 cells are an artificial antigen-presenting cell line that specifically expresses the human major histocompatibility complex class I molecule, HLA-A2. This cell line possesses unique antigen-presenting characteristics; only high-affinity epitope peptides can be effectively presented, forming a stable peptide-MHC (pMHC) complex on the cell surface. Based on this characteristic, T2-A2 cells are widely used in T cell epitope screening and functional validation studies.

[0035] In the T-cell immune response, isolated antigenic epitope peptides cannot directly activate T cells; they must be presented to T-cell receptors via antigen-presenting cells (such as T2-A2 cells) in the form of pMHC complexes. In this study, monkeypox virus-specific T-cell antigenic epitope peptides were identified and loaded onto the surface of T2-A2 cells, successfully constructing an antigenic peptide-antigen-presenting cell complex. Experimental results showed that these epitope peptides effectively activated CD8+ T cells in the peripheral blood of healthy individuals, exhibiting significant immunogenicity. More importantly, the activated T cells demonstrated a strong killing ability against target cells expressing monkeypox virus antigens.

[0036] To further validate the immunological properties of these epitope peptides, researchers assembled them into tetrameric complexes labeled with PE fluorescence. Notably, due to the 96.3% high homology between the monkeypox virus and smallpox virus genomes, and the complete conservation of the two epitope peptide sequences identified in this study in both viruses, these tetramers also showed a specific T-cell response in the peripheral blood of smallpox vaccine recipients. This finding not only confirms that these monkeypox virus CD8+ T-cell epitope peptides can effectively induce T-cell immune responses, but more importantly, their highly conserved nature can prevent immune escape caused by viral mutations, demonstrating great potential as universal vaccine candidate molecules or immunotherapeutic agents.

[0037] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0038] Example 1: Prediction of HLA-A2-restricted epitope peptides of monkeypox virus

[0039] HLA-A2-restricted epitopes were predicted using the MHC-I class molecular prediction tool provided by the US NIH Epitope Database (IEDB) (http: / / tools.iedb.org / mhci / ). Monkeypox virus strain West African clade IIb (ON563414.3) was selected for epitope protein prediction. Finally, two candidate monkeypox virus-specific HLA-A2+ CD8+ epitopes were obtained. + T-cell antigenic epitope peptides are shown in Table 1.

[0040] Table 1. Two antigenic epitopes of monkeypox virus T cells

[0041] .

[0042] Example 2: Identification of HLA-A2 restriction epitope peptides of monkeypox virus

[0043] Example 1: Artificial Synthesis. The predicted candidate T-cell antigen epitope peptide (Nanjing Genscript Biotech Co., Ltd.) was prepared at a concentration of 20 µM. Logarithmically growing T2-A2 cells (T2-A2 was donated by Dr. Anna Gil of the University of Massachusetts Medical School) were seeded into 96-well plates, 10 cells per well. 5The assay was performed using a variety of wells, including blank wells, negative control peptides (EB virus, SEQ ID No. 3, IVTDFSVIK), positive control peptides (influenza A M1 peptide: SEQ ID No. 4, GILGFVFTL), and various synthetic candidate T-cell antigen epitope peptides. Each group had three replicates, with a final volume of 200 µL. After incubation at 37°C for 4 hours, the cells were centrifuged and washed twice. The cells were then labeled with FITC anti-human HLA-A2 (β2m) antibody and incubated at 4°C in the dark for 30 minutes before analysis by flow cytometry. The experiment was repeated three times.

[0044] The results are as follows Figure 1 As shown in Figure A, the results indicate that both antigenic peptides can be effectively presented to T cells by antigen-presenting cells, suggesting that these peptides are T cell antigenic epitope peptides.

[0045] Example 3 Detection of antigen peptide forming pMHC complex

[0046] The two monkeypox virus T-cell antigenic epitopes predicted in Example 1 were detected using ELISA. The specific procedure is as follows:

[0047] This experiment used 96-well U-shaped plates for the preparation and detection of pMHC complexes. The specific operating steps are as follows: First, at room temperature, 100 µL of 0.5 µg / mL streptavidin solution was added to each well, and the plate was incubated on a horizontal shaker for 16–18 hours. After incubation, the plate was washed three times with dedicated washing buffer provided by BioLegend (Cat#420201), with 200 µL of washing buffer added each time, and the liquid was discarded after standing for 1 minute. Subsequently, 100 µL of dilution buffer (containing 0.5 M Tris pH 8.0, 1 M NaCl, 1% BSA, and 0.2% Tween 20) was added for blocking, and the plate was incubated at room temperature for 30 minutes to reduce nonspecific binding.

[0048] Three control groups were set up in the experiment: 1) an HLA blank control, consisting of a pMHC complex formed by the photosensitizing peptide (SEQ ID No. 5: KILGFVFJV) and an MHC molecule (BioLegend, Cat#280003); 2) a positive control, using the influenza A M1 peptide (SEQ ID No. 4, GILGFVFTL), which is known to have good binding activity; and 3) a negative control, using the EB virus peptide (SEQ ID No. 3, IVTDFSVIK). The experimental group consisted of two monkeypox virus T-cell antigenic epitope peptides identified in Example 1.

[0049] In the experimental procedure, 20 µL of a 400 µM antigen peptide solution (pre-treated with a 365 nm triple-use UV analyzer, Qilin Bell Cat#1903274) was first mixed with 20 µL of a photosensitizing peptide pMHC complex (200 µg / mL). 100 µL of dilution buffer was added to prepare the working solution, which was then poured into a pre-treated 96-well plate. The plate was incubated at 37°C in a 5% CO2 cell culture incubator for 1 hour to allow for complete pMHC complex formation. After incubation, the plate was washed three times with washing buffer.

[0050] Subsequently, 100 µL of diluted HRP-labeled secondary antibody (BioLegend, Cat#280303) was added to each well, and incubation was continued at 37°C for 1 hour. After washing, 100 µL of freshly prepared substrate solution (10.34 mL deionized water, 1.2 mL 0.1 M citrate buffer pH 4.0, 240 µL 40 mM ABTS, and 120 µL hydrogen peroxide solution) was added, and the reaction was carried out at room temperature in the dark for 8 minutes. Finally, 50 µL of stop solution (2% oxalic acid solution) was added to terminate the reaction. Within 30 minutes after the reaction was terminated, the absorbance (OD value) of each well was measured at 450 nm using a microplate reader to evaluate the formation efficiency of the pMHC complex.

[0051] The results are as follows Figure 1 As shown in Figure B, the results indicate that both antigenic peptides can form a pMHC complex.

[0052] Example 4: Monkeypox virus HLA-A2 restricted antigenic epitope peptide activates T cells

[0053] T2 cells activate themselves by expressing HLA-A2 molecules (T2-A2, PMID: 34414379; PMID: 35194575; PMID: 35116022; PMID: 37117789). Mononuclear lymphocytes (PBMCs) were isolated from peripheral venous blood of healthy volunteers, and CD8+ T cells were further isolated. T2-A2 cells were labeled with CFSE, treated with 20 μg / mL mitomycin C for 20 min, and then incubated with two different antigenic peptides from Example 1.

[0054] The specific steps are as follows:

[0055] Plant 0.5 × 10⁻⁶ seeds in each well of the 96-well plate. 6 Each CD8+ T cell was loaded with 0.5 × 10⁻⁶ antigenic epitope peptides from the two antigenic epitopes listed in Table 1. 6Two T2-A2 cells were co-cultured (two wells in total) and co-stimulated with 1 µg / mL anti-human CD28 antibody and 50 IU / mL IL-2. 50 IU / mL IL-2 and 20 µM epitope peptide were added every two days.

[0056] CD8+ T cells were isolated from peripheral venous blood of healthy volunteers and co-cultured with T2 cells loaded with antigenic peptides. 1 µg / mL anti-human CD28 antibody and 50 IU / mL IL-2 were added for co-stimulation. After 16 hours of culture, the expression of T cell activation molecules CD69 and CD137 was detected.

[0057] 30 µL of the pMHC complex monomer of the antigenic epitope peptide obtained in Example 3 was mixed with 3.3 µL of PE streptavidin (BioLegend Cat#405203, US) in a 96-well plate and incubated at 4°C in the dark for 30 minutes. Then, 2.4 µL of blocking solution (1.6 µL of 50 mM biotin (Thermo Fisher, Cat#B20656, US)) and 198.4 µL of PBS were added to stop the reaction, and the plate was incubated overnight at 4–8°C to obtain the pMHC complex with the PE fluorescent channel.

[0058] After 7 days of culture, the proportion of specific CD8+ T cells and the percentage of the apoptosis marker Annexin V-APC on T2-A2 cells were labeled with tetramers containing PE fluorescent channels.

[0059] The results are as follows Figure 3 As shown in A and 3B, both monkeypox virus T-cell antigenic epitope peptides predicted in Example 1 can activate T cells. Specifically, the T-cell antigenic epitope peptide LLPSSTAPV corresponding to M1 and the T-cell antigenic epitope peptide SIFLIITKV corresponding to M2 can both activate CD8+ T cells. Furthermore, the specific CD8+ T cells activated by these two peptides can kill target cells, such as… Figure 4 As shown in A and 4B.

[0060] Example 5: Activation of T cells by monkeypox virus HLA-A2 restricted antigenic epitope peptide

[0061] Since monkeypox virus and smallpox virus share a high degree of homology (96.3%), and the two epitope peptide sequences identified in this application are completely conserved in both viruses (100% similarity to smallpox virus sequences), PBMCs were isolated from peripheral venous blood of volunteers 20 years after smallpox vaccination in the absence of monkeypox patients. They were stained with the PE fluorescent channel tetramer and CD8-APC antibody described in Example 1, and then detected by flow cytometry.

[0062] The results are as follows Figure 5As shown in Figures A and 5B, the results indicate that the fluorescently charged tetramers of the two antigenic epitope peptides, M1 and M2, can recognize antigen-specific CD8+ T cells produced in smallpox vaccine recipients. Therefore, these tetramers also showed a specific T cell response in the peripheral blood of smallpox vaccine recipients. This finding not only confirms that these monkeypox virus CD8+ T cell epitope peptides can effectively induce T cell immune responses, but more importantly, their highly conserved nature prevents immune escape due to viral mutations, demonstrating great potential as universal vaccine candidate molecules or immunotherapeutic agents.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A monkeypox virus antigenic epitope peptide, characterized in that: Its amino acid sequence is shown in SEQ ID No.

2.

2. A nucleic acid molecule encoding the antigenic epitope peptide of claim 1.

3. A pMHC complex containing the antigenic epitope peptide of claim 1, characterized in that: The pMHC complex is obtained by refolding MHC monomers and the antigenic epitope peptide described in claim 1; the MHC monomers and the antigenic epitope peptides are mixed in equal volumes; before mixing, the concentration of the MHC monomers is 200 μg / mL and the concentration of the antigenic epitope peptides is 400 μM; the MHC monomers are HLA-A2 heavy chain and HLA-A2 light chain β2m protein.

4. An antigen peptide-antigen presenting cell complex, characterized in that: The antigen-presenting cells are those that have surface-loaded with the antigen epitope peptide of claim 1; the antigen-presenting cells are T2-A2 cells.

5. The use of the antigenic epitope peptide of claim 1, the nucleic acid molecule encoding the antigenic epitope peptide of claim 2, the pMHC complex of claim 3, and / or the antigenic peptide-antigen presenting cell complex of claim 4 in the preparation of a monkeypox virus vaccine or in a medicament for evaluating the efficacy of monkeypox virus vaccination.

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