Above pox virus antigen epitope peptide and application thereof

Abstract

The invention belongs to the technical field of immunotherapy, and particularly relates to a monkey pox virus antigen epitope peptide and application thereof. The invention aims to solve the technical problem that at present, a T cell antigen epitope peptide for universal vaccines of monkey pox viruses is not developed in the field of monkey pox viruses. According to the technical scheme of the invention, the amino acid sequence of the monkey pox virus antigen epitope peptide is shown as SEQ ID No.2. The antigen epitope peptide provided by the invention has very strong immunogenicity, and can induce antigen-specific CD8 + T cells; the antibody can be directly loaded to antigen presenting cells, can activate T cells and effectively induce T cell immunity, and can be used for research and development and preparation of universal vaccines for monkey pox viruses, research and development of drugs and clinical treatment.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of immunotherapy, and particularly relates to a monkeypox virus cell antigen epitope peptide and application thereof. BACKGROUND

[0002] Monkeypox is a zoonotic viral disease caused by the monkeypox virus (MPXV), which belongs to the orthopoxvirus of the poxvirus family. MPXV was first discovered on research monkeys shipped from Singapore in 1958, which may be the reason why the disease is called "monkeypox". However, the natural host of MPXV is more likely to be rodents and other small mammals. The orthopoxvirus genus also includes the variola virus (VARV), and the symptoms of human monkeypox are similar to those of smallpox, but the mortality rate is lower. In the 1970s, sporadic cases of human MPXV were first discovered in several African countries, but in the past 20 years, the virus has spread widely across the African continent. Since May 2022, many countries have reported confirmed cases of monkeypox, and China has reported the first case of monkeypox on June 25, 2022, September 6, and September 16, respectively, all of which are imported cases. At the same time, 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 have reported 127,905 confirmed cases, with 283 deaths, making it one of the most serious epidemics of our time.

[0003] Human monkeypox virus is a zoonotic orthopoxvirus that causes similar clinical symptoms to smallpox after infection. The incubation period of monkeypox is 5-21 days, and the infected person usually presents with "flu-like" symptoms such as fever, headache, fatigue, muscle aches, and lymph node enlargement. The submandibular, neck, or groin area develops soft and fixed lymph nodes that are 1-4 cm in diameter. Monkeypox is often misdiagnosed as varicella and smallpox (up to 50% of cases in the Democratic Republic of the Congo). The main difference between human monkeypox and smallpox is that the human monkeypox virus causes lymph node enlargement, often accompanied by fever; varicella-zoster virus lesions are usually superficial in appearance, with irregular borders, and multiple stages of rash can occur simultaneously on any part of the body, and the rash rapidly evolves from macules to scabbing within 24 hours. The central genomic region of orthopoxviruses is mainly composed of some highly conserved essential genes. The central genomic region of monkeypox virus DNA is 101,466 bp, and has 96.3% homology with the corresponding part of the smallpox virus genome.

[0004] In contrast to antibody and CD4+ T cell immune responses, CD8+ T cell immune responses to monkeypox virus have been less well studied. Early studies have focused on detection of CD8+ T cell activation markers and ELISPOT (Enzyme-Linked ImmunoSpot Assay) assays, with little information on CD8+ T cell epitopes. Adaptive immune responses play an important role in protection against monkeypox virus infection, including B cell production of antibodies, CD4+ T cell (helper T cell) helper and effector functions, and CD8+ T cell (cytotoxic T cell) killing of virus-infected cells. In rhesus macaques, vaccinia virus-specific B cell responses contribute to protection from lethal monkeypox virus infection. Epidemiological studies have further demonstrated that vaccinia virus vaccination protects against infection with other poxviruses, including monkeypox virus. Vaccinia virus-specific memory lymphocytes and antibody levels can persist for more than 50 years after vaccination. However, more than 20 years after vaccination, only about 50% of vaccinates have neutralizing antibody titers greater than 1:32, suggesting that protective immunity to smallpox and possibly cross-protective immunity to monkeypox can wane over time. CD4+ T cells, particularly T follicular helper cells (Tfh), play an important role in enhancing differentiation of memory B cells into antibody-secreting cells. Memory CD4+ T cells can persist for 50 years or more after vaccination with vaccinia virus, with a half-life estimated to be 8-15 years. These vaccinia virus-specific CD4+ T cells 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 anti-vaccinia virus antibody titers. In rhesus macaques infected with simian immunodeficiency virus, if CD4+ T cell counts are less than 300 cells / mm 3 , vaccinia virus-specific immunoglobulin G cannot be produced and the host can die upon challenge with monkeypox virus. In contrast, a patient recently infected with monkeypox virus who was receiving antiretroviral therapy for human immunodeficiency virus type 1 had a CD4+ T cell count of more than 700 cells / mm 3 , and did not develop a severe disease outcome. These results suggest that, in addition to antibodies, T cells play an important role in modulating the severity of monkeypox virus. However, more studies are needed to fully understand the role of T cells in the course of monkeypox virus infection.

[0005] Therefore, it is of great scientific significance and clinical application value to study the CD8+ T cell epitope peptide of monkeypox virus and its application. CD8+ T cells recognize the virus antigen peptide (pMHC) presented by major histocompatibility complex class I molecules through their T cell receptors, and then activate and kill virus-infected cells, playing a key role in anti-viral immunity. Although the current vaccinia virus-based vaccine has a certain cross-protection effect on monkeypox virus infection, this protective effect will weaken over time, and the protection efficacy in different immune status populations is different. It is particularly noteworthy that monkeypox virus is a large DNA virus, and its genome encodes a large number of proteins that may be involved in immune escape, which makes it possible that relying solely on antibody-mediated humoral immunity may not be enough to provide complete protection. Therefore, identifying conserved antigen epitope peptides that can effectively activate CD8+ T cells is crucial for the development of new monkeypox vaccines and immunotherapy strategies. The identification of these epitope peptides not only helps to design universal vaccines that can induce long-lasting T cell immunity, overcoming the challenges posed by potential viral mutations, but also provides additional protection for immunodeficient populations. In addition, T cell immunotherapy based on these epitope peptides, such as polypeptide vaccines or adoptive T cell therapy, is expected to complement traditional antibody therapy, especially in cases where antibody responses are insufficient to provide key protection. 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 immune protection, and ultimately promoting the establishment of more effective monkeypox prevention and control strategies. SUMMARY

[0006] The technical problem to be solved by the present application is that there is currently no T cell antigen epitope peptide for a universal vaccine for monkeypox virus in the field of monkeypox virus.

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

[0008] Further, the present application also provides a nucleic acid molecule encoding the antigen epitope peptide.

[0009] The present application also provides a pMHC complex containing the antigen epitope peptide.

[0010] Further, the pMHC complex is obtained by renaturation of the MHC monomer and the antigen epitope peptide.

[0011] Wherein, the MHC monomer and the antigen epitope peptide are mixed in equal volumes.

[0012] Further, the concentration of the MHC monomer before mixing is 200 μg / mL, and the concentration of the antigen epitope peptide before mixing is 400 μM.

[0013] The application further provides an antigen epitope peptide-antigen presenting cell complex, which is an antigen presenting cell loaded with the antigen epitope peptide.

[0014] Preferably, the CD8+ T cell is a T2-A2 cell.

[0015] Preferably, the CD8+ T cell is a T2-A2 cell.

[0016] Preferably, the CD8+ T cell is a T2-A2 cell.

[0017] Preferably, the CD8+ T cell is a T2-A2 cell.

[0018] The application further provides use of the antigen epitope peptide, the nucleic acid molecule encoding the antigen epitope peptide, the pMHC complex and / or the antigen peptide-antigen presenting cell complex in preparation of a monkeypox virus drug.

[0019] The application further provides use of the antigen epitope peptide, the nucleic acid molecule encoding the antigen epitope peptide, the pMHC complex and / or the antigen peptide-antigen presenting cell complex in screening of a monkeypox virus drug.

[0020] The application further provides use of the antigen epitope peptide, the nucleic acid molecule encoding the antigen epitope peptide, the pMHC complex and / or the antigen peptide-antigen presenting cell complex in preparation of a monkeypox virus vaccine.

[0021] The application further provides use of the antigen epitope peptide, the nucleic acid molecule encoding the antigen epitope peptide, the pMHC complex and / or the antigen peptide-antigen presenting cell complex in preparation of a drug for evaluating the vaccination effect of a monkeypox virus vaccine.

[0022] The application provides a monkeypox virus T cell antigen epitope peptide, which has strong immunogenicity and can induce antigen-specific CD8+ T cells, can be assembled into a pMHC complex with an HLA-A2 heavy chain and an HLA-A2 light chain β2m protein, or can be directly loaded into an antigen presenting cell to activate T cells, effectively induce T cell immunity, avoid immune escape of a monkeypox virus mutant strain, and can detect specific CD8+ T cells in monkeypox virus convalescents, and can be used for monkeypox virus universal vaccine research and development, preparation and drug research and development, and clinical treatment.

[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: 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.

[0024] 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.

[0025] 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.

[0026] 4) Monitoring disease condition: It can be used to monitor changes in the condition of close contacts, medical observers, and suspected and confirmed patients.

[0027] 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

[0028] 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).

[0029] Figure 2Identification of immunogenicity of monkeypox virus T cell epitopes. A: expression level of CD69 after 16 hours; B: expression level of CD137 after 16 hours.

[0030] Figure 3 Detection of antigen-specific T cell generation after stimulation of antigenic epitope peptides; A: antigen-specific T cell generation can be detected after 7 days of stimulation of healthy human CD8+ T cells with fluorescent channel tetramer, B is the statistical chart of A.

[0031] Figure 4 Proportion of T2 cell apoptosis mediated by epitope-stimulated T cells. A: percentage of apoptosis of T2A2 cells stimulated for 7 days, B is the statistical chart of A.

[0032] Figure 5 Evaluation of the proportion of specific CD8+ T cells in vivo 20 years after smallpox vaccination; A: flow cytometry detection of specific CD8+ T cells of the above two (T cell activation can produce antigen-specific CD8+ T epitopes) peptides in HLA-A2+ smallpox vaccinees, B is the statistical chart of A. DETAILED DESCRIPTION

[0033] T2-A2 cells are an artificial antigen-presenting cell line that specifically expresses human major histocompatibility complex class I molecule HLA-A2. This cell line has a unique antigen presentation property, only antigenic epitope peptides with high affinity can be effectively presented by it, and stable peptide-MHC (pMHC) complexes are formed on the cell surface. Based on this property, T2-A2 cells are widely used in T cell epitope screening and functional verification research.

[0034] In the process of T cell immune response, single antigenic epitope peptide cannot directly activate T cells, and must be presented to T cell receptor in the form of pMHC complex by antigen-presenting cells (such as T2-A2 cells). In this study, the obtained monkeypox virus-specific T cell antigenic epitope peptides were loaded on the surface of T2-A2 cells, and an antigen peptide-antigen presenting cell complex was successfully constructed. The experimental results show that these epitope peptides can effectively activate CD8+ T cells in healthy human peripheral blood, showing significant immunogenicity. More importantly, the activated T cells exhibit strong killing ability to target cells expressing monkeypox virus antigens.

[0035] To further verify the immunological properties of these epitope peptides, researchers assembled them into tetramer complexes with PE fluorescent labeling. Notably, due to the high homology of 96.3% between the monkeypox virus and the smallpox virus genome, and the complete conservation of the two epitope peptide sequences identified in this study in both viruses, these tetramers can also detect specific T cell responses in the peripheral blood of smallpox vaccinees. 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 avoid immune escape caused by viral mutations, showing great potential as a universal vaccine candidate molecule or immunotherapy preparation.

[0036] The present application will be further described in conjunction with the drawings and specific embodiments of the present application, which are used to explain the present application and are not intended to limit the scope of the present application. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0037] Example 1 Prediction of monkeypox virus HLA-A2 restricted antigen epitope peptides HLA-A2 restricted antigen epitope prediction was performed using the MHC-I class molecule prediction tool provided by the NIH antigen epitope database (IEDB) (http: / / tools.iedb.org / mhci / ). The monkeypox virus West African branch IIb (ON563414.3) strain was selected for antigen epitope protein prediction. Finally, two candidate monkeypox virus specific HLA-A2+ CD8 + T cell epitope peptides were obtained, as shown in Table 1.

[0038] Table 1 Monkeypox virus T cell 2 antigen epitope peptides Example 2 Identification of monkeypox virus HLA-A2 restricted antigen epitope peptides The candidate T cell antigen epitope peptides predicted in Example 1 were artificially synthesized (Nanjing Kingsriver Biotechnology Co., Ltd.), and were configured at a concentration of 20 µM. Logarithmic growth state T2-A2 cells (T2-A2 was a gift from Dr. Anna Gil, University of Massachusetts Medical School) were planted into a 96-well plate at 10 5Each well was set with 3 holes, and each group of holes was set with blank holes, negative control peptide (EB virus, SEQ ID No. 3, IVTDFSVIK), positive control peptide (influenza A M1 peptide: SEQ ID No. 4, GILGFVFTL) and each synthetic candidate T cell epitope peptide, with a final volume of 200 μL. After incubation at 37°C for 4 hours, centrifugal washing was performed twice, followed by labeling with FITC anti-human HLA-A2 (β2m) antibody, incubation at 4°C for 30 minutes in the dark, and detection by flow cytometry. The experiment was performed for 3 times.

[0039] The results are shown in Figure 3A, which shows that both antigen polypeptides can effectively present information to T cells by antigen presenting cells, indicating that these peptides are T cell epitope peptides. Figure 1

[0040] Example 3 Detection of antigen polypeptide forming pMHC complex The two monkeypox virus T cell epitope peptides predicted in Example 1 were detected by ELISA. The specific operation is as follows: In this experiment, 96-well U-shaped plates were used for the preparation and detection of pMHC complex. The specific operation steps are as follows: first, 100 μL of streptavidin solution with a concentration of 0.5 μg / mL was added to each well under room temperature conditions, and incubated on a horizontal shaker for 16-18 hours. After incubation, the special washing buffer provided by BioLegend company (Cat#420201) was used for washing 3 times, 200 μL of washing solution was added each time, and the liquid was discarded after standing for 1 minute. Then, 100 μL of dilution buffer (containing 0.5M Tris pH 8.0, 1M NaCl, 1% BSA and 0.2% Tween 20) was added for blocking treatment, and incubated at room temperature for 30 minutes to reduce non-specific binding.

[0041] Three control groups were set up in the experiment: 1) HLA blank control, pMHC complex formed by photosensitive peptide (SEQ ID No. 5: KILGFVFJV) and MHC molecule (BioLegend, Cat#280003); 2) positive control, using influenza A M1 peptide (SEQ ID No. 4, GILGFVFTL) with good binding activity; 3) negative control, using EB virus peptide (SEQ ID No. 3, IVTDFSVIK). The experimental group was the two monkeypox virus T cell epitope peptides identified in Example 1.

[0042] ​For experimental operation, first mix 20 μΐ of antigen peptide solution (previously treated with 365 nm tri-purpose ultraviolet analyzer, Qilin Bell Cat#1903274) with a concentration of 400 μΜ with 20 μΐ of photosensitive peptide pMHC complex (200 μg / mL), add 100 μΐ of dilution buffer to prepare working solution, and add to the treated 96-well plate. Place the plate in a 37°C, 5% CO2 cell incubator for 1 hour to allow the pMHC complex to form fully. After incubation, wash again with washing buffer for 3 times.

[0043] Subsequently, add 100 μΐ of diluted HRP-labeled secondary antibody (BioLegend, Cat#280303) to each well, and continue incubation at 37°C for 1 hour. After washing, add 100 μΐ of freshly prepared substrate solution (10.34 mL deionized water, 1.2 mL 0.1M citric acid buffer pH 4.0, 240 μΐ 40 mM ABTS and 120 μΐ hydrogen peroxide solution), and react at room temperature for 8 minutes in the dark. Finally, add 50 μΐ of stop solution (2% oxalic acid solution) to terminate the reaction. Within 30 minutes after the reaction is terminated, use a microplate reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm to evaluate the formation efficiency of the pMHC complex.

[0044] The results are shown in FIG. 6B, which show that both antigen polypeptides can form pMHC complexes. Figure 1

[0045] Example 4 Activation of T cells by monkeypox virus HLA-A2 restricted antigen epitope peptides T2 cells activate T cells by expressing HLA-A2 molecules (T2-A2, PMID: 34414379; PMID: 35194575; PMID: 35116022; PMID: 37117789). Single nuclear lymphocytes (PBMCs) in the peripheral venous blood of healthy volunteers are isolated and further separated into CD8+ T cells. T2-A2 cells are labeled with CFSE, and after being treated with 20 μg / mL mitomycin for 20 minutes, they are incubated with the two different antigen peptides in Example 1, respectively.

[0046] Specifically: 0.5 x 10 6 CD8+ T cells are co-cultured with 0.5 x 10 6 T2-A2 cells loaded with 11 antigen epitope peptides in Table 1 (a total of 11 wells), and are co-stimulated with 1 μg / mL anti-human CD28 antibody and 50 IU / mL IL-2. 50 IU / mL IL-2 and 20 μΜ antigen epitope peptides are supplemented every two days.​

[0047] Isolate CD8+T cells from peripheral vein blood of healthy volunteers, and co-culture with T2 cells loaded with antigen polypeptides, add 1 μg / mL anti-human CD28 antibody and 50 IU / mL IL-2 for co-stimulation, detect the expression of T cell activation molecules CD69 and CD137 after 16 hours of culture.

[0048] Mix 30 μL of pMHC complex monomer of the antigen epitope peptide obtained in Example 4 with 3.3 μL of PE streptavidin (BioLegend Cat#405203, US) in a 96-well plate, and after incubation at 4°C in the dark for 30 minutes, add 2.4 μL of blocking solution (1.6 μL of 50 mM biotin (Thermo Fisher, Cat#B20656, US) and 198.4 μL of PBS to stop the reaction, and incubate overnight at 4-8°C to obtain pMHC complex with PE fluorescence channel.

[0049] Label the proportion of specific CD8+T cells and the percentage of Annexin V-APC apoptosis marker on T2-A2 cells with PE fluorescence channel tetramer after 7 days of culture.

[0050] The results are shown in Figure 3 As shown in Figures 1A and 1B, the two monkeypox virus T cell antigen epitope peptides predicted in Example 1 can activate T cells. Among them, the T cell antigen epitope peptide LLPSSTAPV corresponding to M1 and the T cell antigen epitope peptide SIFLIITKV corresponding to M2 can activate CD8+T cells. At the same time, the specific CD8+T cells activated by the above two peptides can kill target cells, as shown in Figure 4 Figures 2A and 2B.

[0051] Example 5 Activation of T cells by monkeypox virus HLA-A2 restricted antigen epitope peptides Since the monkeypox virus has a high homology of 96.3% with the smallpox virus genome, and the two epitope peptide sequences identified in the present application are completely conserved in the two viruses (100% similarity with the smallpox virus sequence); therefore, in the absence of monkeypox patients, PBMCs isolated from peripheral vein blood of volunteers 20 years after smallpox vaccination were stained with PE fluorescence channel tetramer and CD8-APC antibody in Example 1, and then detected by flow cytometry.

[0052] The results are shown in Figure 5As shown in Figures 5A and 5B, the results showed that the fluorescent channel tetramer with Ml and M2 antigen epitope peptides could recognize antigen-specific CD8+T cells in smallpox vaccinees. Therefore, these tetramers could also detect specific T cell responses in the peripheral blood of smallpox vaccinees. This finding not only confirmed that these monkeypox virus CD8+T cell epitope peptides could effectively induce T cell immune responses, but more importantly, their highly conserved nature could avoid immune escape due to viral mutations, showing great potential as a universal vaccine candidate molecule or immunotherapy preparation.

[0053] Finally, it should be noted that the above examples are only intended to illustrate the technical solutions of the present application and not to limit the scope of protection of the present application. For those skilled in the art, on the basis of the above description and ideas, other different forms of changes or modifications can also be made, which do not need or cannot be exhausted here. Any modification, equivalent replacement and improvement made within the spirit and principles of the present application shall be included in the protection scope of the claims of the present application.

Claims

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

2.

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

3. A pMHC complex comprising the antigenic epitope peptide of claim 1.

4. The pMHC complex of claim 3, wherein: The pMHC complex is obtained by renaturation of the MHC monomer and the antigenic epitope peptide of claim 1; mixing equal volumes of the MHC monomer and the antigenic epitope peptide; before mixing, the concentration of the MHC monomer is 200 μg / mL, and the concentration of the antigenic epitope peptide is 400 μM.

5. An antigenic peptide-antigen presenting cell complex, characterized in that: An antigen presenting cell loaded with the antigenic epitope peptide of claim 1.

6. The antigenic peptide-antigen presenting cell complex of claim 5, wherein: The antigen presenting cell is a CD8+ T cell.

7. The antigenic peptide-antigen presenting cell complex according to claim 6, characterized in that: The CD8+ T cell is a T2-A2 cell.

8. 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 or 4, and / or the antigenic peptide-antigen presenting cell complex of any one of claims 5-7 in the preparation of a monkeypox virus drug or in the screening of a monkeypox virus drug.

9. 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 or 4, and / or the antigenic peptide-antigen presenting cell complex of any one of claims 5-7 in the preparation of a monkeypox virus vaccine or in the evaluation of the vaccination effect of a monkeypox virus vaccine.

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