African swine fever virus t cell epitope peptide for inducing porcine t cell immune response and application thereof

Abstract

This invention discloses an African swine fever virus (ASFV) T-cell epitope peptide and its composition for inducing porcine T-cell immune responses. The amino acid sequence is as shown in any of SEQ ID NO. 1-19, or an amino acid sequence derived from SEQ ID NO. 1-19 but retaining the protein function shown in SEQ ID NO. 1-19, through substitution and / or deletion and / or addition of one or more amino acid residues. The composition comprises amino acids with the sequences of SEQ ID NO. 1-19. This invention, combined with IFN-γ ELISpot and flow cytometry screening, yielded a group of key ASFV T-cell antigens and epitope peptides, which can be used as vaccine components to induce strong T-cell immune responses. The T cells activated by these antigenic peptides have the greatest immunoprotective potential and are essential key antigens for ASFV vaccine development.

Description

Technical Field

[0001] This invention belongs to the field of bioscience and diagnostic reagent technology, and relates to the prevention and treatment of animal diseases, specifically to African swine fever virus T cell epitope peptides that induce porcine T cell immune responses and their applications. Background Technology

[0002] African swine fever (ASF) is an acute, hemorrhagic, and highly contagious disease caused by the African swine fever virus (ASFV). It can cause 100% mortality in both domestic and wild pigs, and has become the greatest threat to the pig farming industry. In August 2018, genotype II ASFV was introduced into my country and subsequently spread to Southeast Asia, causing enormous economic losses to the pig farming industry.

[0003] African swine fever virus (ASFV) is the sole member of the genus *African swine fevervirus* within the family African swine feverviridae, and the only known arbovirus-borne DNA virus. Its genome is a 170-194 kb linear double-stranded DNA, encoding over 150 proteins, including 54 structural proteins and over 100 non-structural proteins. Currently, there is no commercially available ASFV vaccine. Identifying and identifying protective antigens for ASFV is crucial for ASFV vaccine development. While existing attenuated ASFV strains are effective, they carry risks such as genetic recombination and virulence reversion. Existing inactivated ASFV vaccines have been shown not to induce immune protection. Subunit vaccines, due to their reliable safety profile, have become an alternative option for ASFV vaccine development.

[0004] Existing research indicates that ASFV relies more heavily on T-cell immune-mediated immune protection. Inactivated ASFV vaccines and subunit vaccines based on the structural proteins p72, p54, and p30, even when inducing neutralizing antibodies, do not provide immune protection against ASFV. However, immunization with naturally attenuated OURT88 / 3 and NH / P68 strains on macrophages and bone marrow-derived dendritic cells induces 100% protection against both homologous and heterologous strains, and this vaccine-induced immune protection is eliminated by using CD8 T cells lacking anti-CD8 antibodies. This suggests that the T-cell immune response induced by attenuated ASFV strains plays a crucial role in immune protection. Using IFN-γ ELISpot technology, Dixon et al. (2019) identified 38 T-cell-recognizable proteins in genotype I ASFV-immunized convalescent pigs. Goatley et al. (2020) further discovered that antigenic components containing p30, p54, p72, pB602L, pE199L, pEP153R, F317L, and MGF505-5R have immunoprotective effects. Portugal et al. (2024) demonstrated that inoculation with six of the above antigens can induce protective immunity against genotype I ASFV. However, the protective antigens and T-cell recognition epitopes of genotype II ASFV are unclear. Although many research institutions at home and abroad have conducted research on protein and peptide vaccines such as p30, p54, and p72, there is still no safe and effective ASF vaccine on the market. Therefore, identifying the ASFV antigens and T-cell epitopes specifically recognized by T cells in surviving pigs challenged with genotype II ASFV can provide a theoretical basis for the development of novel ASFV vaccines. Summary of the Invention

[0005] This invention addresses the technical problem at hand and overcomes the shortcomings of existing technologies by providing ASFV T cell antigens and epitope peptides that are highly immunogenic and can induce porcine-specific T cell responses, screened using IFN-γELISpot technology.

[0006] This invention also provides a method for identifying T cell subsets activated by different T cell epitope peptides of ASFV using flow cytometry (FCM), as well as the functional characteristics of the epitopes.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] The present invention provides an African swine fever virus T cell epitope peptide that induces a porcine T cell immune response, the amino acid sequence of which is shown in any of SEQ ID NO. 1-19, or an amino acid sequence derived from SEQ ID NO. 1-19 and retaining the protein function shown in SEQ ID NO. 1-19 by substitution and / or deletion and / or addition of one or more amino acid residues.

[0009] The present invention also provides a composition of African swine fever virus T cell epitope peptides that induce porcine T cell immune responses, the composition comprising amino acids having the sequence SEQ ID NO.1-19.

[0010] The present invention further provides the application of the African swine fever virus T-cell epitope peptide or its composition in the preparation of African swine fever virus vaccine, wherein the epitope peptide or epitope peptide composition serves as a candidate immunogen for ASFV epitope vector vaccine.

[0011] The further optimized technical solution of this invention is as follows:

[0012] Preferably, the amino acid sequence of SEQ ID NO.1 is recognized by CD4 T cells of ASFV-recovered pigs and contains SLA-II class-restricted T cell epitopes; the amino acid sequences of SEQ ID NO.2-5 are recognized by CD8 T cells of ASFV-recovered pigs and contain SLA-I class-restricted T cell epitopes; the amino acid sequences of SEQ ID NO.6-19 are simultaneously recognized by CD4 and CD8 T cells of ASFV-recovered pigs and contain both SLA-I and -II class-restricted T cell epitopes.

[0013] Preferably, the sequences SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.11 and SEQ ID NO.12 are recognized by cytotoxic T cells (CTLs) of ASFV-recovered pigs and contain SLA-I class-restricted T cell epitopes.

[0014] Preferably, the African swine fever virus T-cell epitope peptides are derived from ASFV. CP204L , A151R , I215L , E184L , EP153R , E146L The gene or encoding protein, and the African swine fever virus T-cell epitope peptide or its derived protein can be used to design antigenic components or combinations of formulations for ASFV vaccines.

[0015] Preferably, the African swine fever virus T-cell epitope peptide or its derived protein is obtained by screening and verification from the peripheral blood of recovered pigs that survived immunization with an attenuated ASFV strain and challenged with a virulent ASFV strain using IFN-γ ELISpot assay, T-cell proliferation assay, intracellular cytokine staining and CD107a surface staining.

[0016] This invention selects the complete amino acid sequence of the potential protective protein antigen of the prevalent Chinese strain of ASFV, and uses peptide synthesis technology to synthesize overlapping peptides covering the entire sequence of each protein. Piglets are immunized with a weakened strain of ASFV, and 29 days after immunization, they are infected with a virulent strain of ASFV. Peripheral blood mononuclear cells (PBMCs) are collected from recovered piglets at 5, 15, and 42 days post-infection. Immunological assays such as IFN-γ ELISpot assay, FCM, and in vitro culture of T cells are used to screen and identify dominant T cell epitope peptides of ASFV, and further analyze the T cell subset types (CD4 T or CD8 T cells) activated by the epitope peptides, providing a theoretical basis for the development of novel ASF subunit vaccines.

[0017] Preferably, the African swine fever virus T cell epitope peptide or its derived protein is co-cultured with peripheral blood of ASFV-recovered pigs for 18 hours, specifically stimulating T cells to secrete IFN-γ (forming spots) in the porcine IFN-γ ELISpot assay; specifically stimulating CD4 and / or CD8 T cells to secrete IFN-γ, TNF-α, or IL-2 in the intracellular cytokine staining assay; specifically stimulating CD4 and CD8 T cell proliferation (dilution occurs in CSFE) by co-culturing with CFSE-labeled peripheral blood of ASFV-recovered pigs for 72 hours in the T cell proliferation assay; and specifically stimulating CD8 T cells to express CD107a on the surface in the CD107a surface staining assay.

[0018] Preferably, the gene encoding the African swine fever virus T-cell epitope peptide or its derived protein is used in the design and application of ASFV subunit vaccines, recombinant vector vaccines, mRNA vaccines, and DC vaccines, and has the potential for immune protection. Whether the above-mentioned antigen components are used in the vaccine formulation can be determined by detecting the presence of T-cell responses specific to each antigen peptide or protein in the corresponding vaccine-immunized pigs using the IFN-γ ELISpot assay, T-cell proliferation assay, intracellular cytokine staining, and CD107a surface staining method.

[0019] Preferably, at least one African swine fever virus T-cell epitope peptide or its derived protein is selected and evaluated using the IFN-γELISpot assay, T-cell proliferation assay, intracellular cytokine staining, and CD107a surface staining method to assess the strength of immune protection and / or T-cell immune response induced by any ASFV vaccine.

[0020] This invention identifies and screens dominant T-cell epitopes of African swine fever virus (ASFV). The epitope peptides screened in this invention are antigens specifically recognized by surviving porcine T cells after ASFV challenge and can serve as candidate immunogens for ASFV epitope vector vaccines. The ELISpot / FCM detection and identification method established in this invention provides a powerful tool for evaluating the protective efficacy of ASFV candidate vaccines and screening and identifying dominant ASFV epitopes, and also provides a theoretical basis for the development of ASFV subunit vaccines.

[0021] The beneficial effects of this invention are as follows: This invention combines IFN-γ ELISpot and FCM technology to screen and obtain a group of key T cell antigens and epitope peptides for ASFV, which can be used as vaccine components to induce a strong T cell immune response. The T cells activated by these antigen peptides have the greatest potential for immune protection and are essential key antigens for ASFV vaccine development.

[0022] This invention combines IFN-γ ELISpot assay, FCM screening, and identification of a group of ASFV highly immunogenic T-cell epitope peptides. It also elucidates the T-cell subtypes activated by these peptides and the functional characteristics of the polypeptides, deepening our understanding of ASFV T-cell epitope peptides. Furthermore, it provides an efficient technical solution for high-throughput screening of ASFV T-cell and B-cell epitopes, enriching the variety of ASFV T-cell epitope peptides and greatly advancing the research of ASF subunit vaccines. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the ASFV T cell epitope peptide screening assay of the present invention.

[0024] Figure 2 This is a dynamic graph showing the frequency of peptide-specific T cells in the peptide pool at different time points after immunized pigs were challenged with the virus according to the present invention.

[0025] Figure 3 This is a schematic diagram illustrating the cross-multiplication method used in this invention to determine the epitope peptide that induces specific T cells to secrete IFN-γ.

[0026] Figure 4 This is a schematic diagram illustrating the results of the preliminary screening of positive T-cell epitope peptides for the IFN-γ ELISpot validation of this invention.

[0027] Figure 5 This is a schematic diagram illustrating the results of the ICS identification of T cell subtypes activated by T cell epitope peptides in this invention.

[0028] Figure 6 This diagram illustrates the results of the proliferation assay of the present invention to identify the ASFV T cell epitope-induced proliferation of CD4 T and CD8 T cells.

[0029] Figure 7This is a schematic diagram illustrating the results of the FCM assay used in this invention to identify cytotoxic T-cell epitope peptides. Detailed Implementation

[0030] The technical solution of the present invention will be further described in detail below with reference to the embodiments: This embodiment is implemented under the premise of the technical solution of the present invention, and provides detailed implementation methods and specific operation processes, but the protection scope of the present invention is not limited to the following embodiments.

[0031] The materials and reagents mentioned in this invention are all available to the public through domestic and international commercial channels or free channels.

[0032] Example 1: Screening for T cell dominant epitope peptides

[0033] 1. ASFV polypeptide synthesis

[0034] Based on the amino acid sequences of pA151R, pE184L, pE199L, pI215L, pE146L, pI8L, and pEP153R of the prevalent Chinese strain ASFV CADC_HN09 (GenBank entry number: MZ614662), overlapping polypeptides of the proteins were synthesized by Nanjing GenScript Biotech Co., Ltd. Each polypeptide contains 20 amino acids, with adjacent polypeptides containing 10 overlapping amino acids. Potential epitopes in the p72, p54, pB602L, pMGF505-4R, and pM448R proteins that may be simultaneously recognized by MHC class I and II molecules were predicted and synthesized using the IEDB website. All polypeptides were then classified according to... Figure 3 The peptides were arranged as shown and diluted with sterile PBS to prepare solutions of single peptides at a concentration of 5 mg / mL. 20 μL of 10 single peptide solutions from each column were mixed to form one peptide pool, and 20 μL of 14 single peptide solutions from each row were mixed to form another peptide pool, and so on, to prepare a total of 24 peptide pools.

[0035] 2. ASFV attenuated strain immunization and challenge protection test

[0036] Eight healthy 6-week-old piglets were randomly divided into two groups: a group of five piglets immunized with an attenuated ASFV strain and a control group of three piglets. The piglets were immunized with the attenuated ASFV CADC_HN09 strain via intramuscular injection into the neck, with a dosage of 10 mg / L. 6 One ASFV / head, with no immunization in the control group. 29 days post-immunization, both the immunized and control groups received an intramuscular injection of 10 HAD. 50 A high dose of the virulent ASFV CADC_HN09 strain was administered. Anticoagulated blood was collected from the immune challenge group at 5, 15, and 42 days post-challenge, and 5 days post-second challenge (47 days post-first challenge for the second challenge). PBMCs were isolated for IFN-γ ELISpot and FCM identification. The experimental procedure is as follows: Figure 1As shown in the figure. Clinical symptoms were continuously observed after challenge. It was found that all pigs in the control group died approximately 10 days after challenge, indicating successful challenge. All pigs in the immunized challenge group survived the entire experiment without significant fever or other clinical symptoms, indicating that the attenuated strain induced a good protective effect.

[0037] 3. Isolation of pig PBMCs

[0038] At each sampling time point described in step 2, venous blood was aseptically collected from the test pigs using negative pressure anticoagulant tubes. Porcine peripheral blood mononuclear cells (PBMCs) were isolated using a porcine peripheral blood mononuclear cell isolation kit (Tianjin Haoyang Biotechnology Co., Ltd.). Specific steps are detailed in the manufacturer's instructions. Red blood cells in the PBMCs were lysed using erythrocyte lysis buffer. Viable cells were counted using a hematology counter after trypan blue staining.

[0039] 4. IFN-γ ELISpot assay for identifying T cell epitope peptides

[0040] Add the PBMC single-cell suspension prepared in step 3 above to each well of the ELISpot 96-well plate, 2×10 5 Peptide pools or single peptide solutions were added to each well, and the plate was incubated at 37°C in a 5% CO2 cell culture incubator for 18 h. The plate was removed, the supernatant was discarded, and the plate was washed with sterile distilled water. Biotin-labeled anti-pig IFN-γ antibody (P2C11-Biotin, Mabtech, Sweden) was added and incubated for 2 h. After washing with PBS, the secondary antibody Streptavidin-ALP (Mabtech, Sweden) was added and incubated for 1 h. The washing steps were repeated. The substrate BCIP / NBT-plus (Mabtech, Sweden) was added, and the plate was allowed to stand until spots appeared. The liquid in the wells was discarded, and the plate was washed with ultrapure water. The plate was air-dried, and the number of spots was counted using an ImmunoSpot Analyzer ELISpot (CTL, USA). The results are as follows: Figure 2 As shown, peptide pools 1, 4, 5, 11, 12, 14, 16, 17, 19, 20, 22, and 24 were identified by two or more pigs and induced the most specific T cells, thus forming strongly positive peptide pools (all ELISpot results have been adjusted for natural background). Figure 2 In the diagram, A represents the dynamic frequency of peptide-specific T cells 5 days after infection; B represents the dynamic frequency of peptide-specific T cells 15 days after infection; and C represents the dynamic frequency of peptide-specific T cells 42 days after infection. The dashed line indicates a strong positive boundary. Figure 3 As shown, using the cross-multiplication method, 40 strongly positive epitope peptides were initially screened. Figure 4 As shown, different strongly positive peptides were used to stimulate PBMCs of recovered pigs to perform the IFN-γ ELISpot test, further verifying... Figure 3 The result. Figure 4The top-middle figure shows the identification results of epitope peptides in convalescent pig No. 1; the bottom figure shows the identification results of epitope peptides in convalescent pig No. 2. A total of 19 strongly positive T-cell epitope peptides were identified: pA151R-P6, pA151R-P7, pA151R-P9, pE184L-P1, pE184L-P4, pE184L-P6, pE184L-P7, pE184L-P10, pI215L-P9, pI215L-P11, pI215L-P12, pI215L-P18, pI215L-P20, and pEP153R-P10 (see Table 1).

[0041] Table 1. ASFV T cell epitope peptide sequences and functional characteristics

[0042]

[0043] Note: + indicates a positive test; - indicates a negative test; N indicates no test was conducted.

[0044] Example 2: Identification of T cell subtypes activated by T cell epitope peptides using the ICS assay

[0045] 1. Peptide stimulation

[0046] Two PBMCs from recovered pigs were seeded into 96-well U-shaped plates. Nineteen single peptide solutions (5 mg / mL) dissolved in PBS were added to each well, with two replicates. The final peptide concentration was 5 μg / mL. The plates were incubated in a cell culture incubator for 18 h.

[0047] 2. Flow cytometry (FCM) surface staining

[0048] Add primary antibody mixture [flow cytometry antibody (BD, USA) containing anti-porcine CD3ε, CD4, CD8α, and TCRγδ molecules] to each well, resuspend cells, and incubate for 25 min. Add PBS solution containing 0.5% BSA, centrifuge, and discard the supernatant. Fix cells with 4% PFA.

[0049] 3. ICS (Intracellular Cytokine Staining)

[0050] Cell membranes were perforated for 15 min using Perm / Wash perforation buffer (BD, USA), and then incubated for 25 min with IFN-γ antibody solution (IFN-γ antibody prepared with Perm / Wash buffer). The antibody solution was washed away with Perm / Wash, and cells were fixed with 0.5% PFA. Flow cytometry was then used to detect IFN-γ expression in CD4 T and CD8 T cells stimulated with a single peptide. Figure 5The left image shows an epitope peptide that can stimulate CD4 T cells to secrete IFN-γ; the right image shows an epitope peptide that can stimulate CD8 T cells to secrete IFN-γ; 1640 in the image represents the negative control cultured in 1640 medium; PMA / Iono represents the positive control. Results are as follows: Figure 5 As shown, pI215L-P12 can only stimulate CD4 T cells to secrete IFN-γ and contains SLA-II restricted T cell epitopes. Four peptides, pA151R-P1, pE184L-P2, and pI215L-P13 / P18, can only induce CD8 T cells to secrete IFN-γ and contain SLA-I restricted T cell epitopes. However, 14 peptides, pA151R-P6 / P7 / P9, pE184L-P2 / P4, pI215L-P9 / P11 / P13 / P18 / P20, pE146L-P8 / 12, and pEP153R-P2 / P3, can simultaneously activate both CD4 and CD8 T cells and contain both SLA-I and SLA-II restricted T cell epitopes.

[0051] Example 3: Cell proliferation assay to identify epitope peptides that can stimulate the proliferation of CD4 T and CD8 T cells

[0052] 1. CFSE mark

[0053] Take convalescent pig PBMCs, resuspend them in an appropriate amount of 37°C CFSE staining solution, and mix gently. Incubate at 37°C in the dark. Then add complete culture medium to stop the labeling. Centrifuge and discard the supernatant. Resuspend the cells in an appropriate amount of complete culture medium and seed them into 96-well U-shaped plates. Add peptide solution to each well to a final concentration of 5 μg / mL, and incubate in a cell culture incubator for 3 days.

[0054] 2. FCM identification

[0055] Incubate primary antibody (flow cytometry antibody containing anti-porcine CD3ε, CD4, CD8α, and TCRγδ molecules): The specific steps are the same as those for the flow cytometry surface staining described above. Figure 6 The left panel shows epitope peptides promoting CD4 T cell proliferation; the right panel shows epitope peptides promoting CD8 T cell proliferation; 1640 represents the negative control; weakly positive CFSE indicates proliferating T cells. Results are as follows. Figure 6 As shown, pI215L-P12 can only induce CD4 T cell proliferation and contains CD4 T cell epitopes; the four polypeptides pA151R-P1, pE184L-P2 / P4, and pI215L-P18 can only induce CD8 T cell proliferation and contain CD8 T cell epitopes; p30-P14, pA151R-P6 / P7 / P9, pE184L-P1 / P7, and pI215L-P11 can induce both CD4 and CD8 T cell proliferation simultaneously. These results corroborate those of Example 2.

[0056] Example 4 Identification of cytotoxic T-cell epitope peptides of ASFV

[0057] To further identify CTL epitope peptides among CD8 T cell epitope peptides, PBMCs from two recovered pigs were seeded into 96-well U-shaped plates. Peptides recognized by CD8 T cells were added to the wells, along with CD107a antibody (Bio-rad, USA). The plates were incubated for 18 h. Then, flow cytometry surface staining was performed as in Example 2. Figure 7 #1 represents the result of testing on convalescent pig #1; #2 represents the result of testing on convalescent pig #2; 1640 is the negative control. Results are as follows: Figure 7 As shown, a total of 7 peptides, including pA151R-P1 / P7, pE184L-P1 / P7, and pI215L-P9 / P12 / P13, can induce CD8αβ T cells (CTLs) to express the CD107a molecule, which is a cytotoxic T cell epitope peptide.

[0058] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any transformations or substitutions that can be conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. An African swine fever virus T-cell epitope peptide that induces a porcine T-cell immune response, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

2. The composition of African swine fever virus T-cell epitope peptides for inducing porcine T-cell immune responses according to claim 1, characterized in that, The composition comprises amino acids with the sequence SEQ ID NO.1-19.

3. The use of the composition of the African swine fever virus T-cell epitope peptide as described in claim 1 or the African swine fever virus T-cell epitope peptide as described in claim 2 in the preparation of an African swine fever virus vaccine, characterized in that, The epitope peptide or a composition of epitope peptides serves as a candidate immunogen for an ASFV epitope vector vaccine.

4. The application according to claim 3, characterized in that, The African swine fever virus T-cell epitope peptides or the composition of epitope peptides are respectively derived from ASFV. CP204L , A151R , I215L , E184L , EP153R , E146L The gene or encoding protein, and the composition of the African swine fever virus T-cell epitope peptide and epitope peptide is used to prepare an ASFV vaccine.

5. The method for preparing African swine fever virus T-cell epitope peptides as described in claim 1, characterized in that, The African swine fever virus T-cell epitope peptides were screened and verified from the peripheral blood of recovered pigs that survived immunization with an attenuated ASFV strain and challenged with a virulent ASFV strain using the IFN-γ ELISpot assay, T-cell proliferation assay, intracellular cytokine staining, and CD107a surface staining method.

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