Antigen epitope peptide combination of broad-spectrum influenza vaccine and application of antigen epitope peptide combination

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to an antigen epitope peptide composition of a broad-spectrum influenza vaccine and application of the antigen epitope peptide composition. The antigen epitope peptide combination disclosed by the invention comprises an H1N1 antigen epitope peptide combination and an H5N1 antigen epitope peptide combination. The H1N1 antigen epitope peptide combination disclosed by the invention comprises eight types of antigen epitopes recognized by an influenza broad-spectrum neutralizing antibody; the H5N1 antigen epitope peptide combination comprises another four types of antigen epitopes recognized by influenza broad-spectrum neutralizing antibodies, and the antigen epitope peptide combination can induce a host to generate broad-spectrum neutralizing antibody response and is used for preparing broad-spectrum influenza vaccines. Results of the embodiment show that compared with a blank control group and a trivalent influenza vaccine group, the mouse virus load of the prepared Ag3 + Ag4 influenza vaccine is reduced by 10-84 times.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to an antigen epitope peptide combination of a broad-spectrum influenza vaccine and an application thereof. Background Art

[0002] Influenza viruses pose a serious threat to global public health each year. Although seasonal influenza vaccines are widely used for influenza prevention and control, due to the continuous antigenic drift and shift of the virus, existing vaccines often need to be updated annually to respond to the different circulating virus strains. This compatibility limitation significantly affects the protective efficacy of seasonal influenza vaccines. Therefore, the development of an influenza vaccine with broad-spectrum protection that can provide immune protection against multiple influenza strains without relying on specific virus strains has become an urgent scientific challenge.

[0003] The hemagglutinin (HA) protein, one of the most important structural proteins on the surface of the influenza virus, consists of a highly variable head and a relatively conserved stem. Neutralizing antibodies induced by certain antigenic epitope regions of the HA protein can achieve broad-spectrum neutralization independent of influenza variants, preventing the virus from invading host cells. However, current immunization strategies targeting these conserved epitopes are still inadequate, and there is a lack of influenza vaccines that can accurately induce multiple broad-spectrum neutralizing antibody responses. Therefore, developing an influenza vaccine that can simultaneously and accurately induce multiple broad-spectrum neutralizing antibody responses remains a major challenge in influenza vaccine research and development. Summary of the Invention

[0004] The purpose of the present invention is to provide an antigenic epitope peptide combination for a broad-spectrum influenza vaccine and its application. The antigenic epitope peptide combination of the present invention can induce a variety of broad-spectrum influenza neutralizing antibody responses and can be used to prepare a broad-spectrum influenza vaccine.

[0005] The present invention provides an antigen epitope peptide combination for a broad-spectrum influenza vaccine, wherein the antigen epitope peptide combination comprises an H1N1 antigen epitope peptide combination and an H5N1 antigen epitope peptide combination; the H1N1 antigen epitope peptide combination comprises a first antigen epitope peptide to a fifteenth antigen epitope peptide; the H5N1 antigen epitope peptide combination comprises a sixteenth antigen epitope peptide to a twenty-sixth antigen epitope peptide; the amino acid sequences of the first antigen epitope peptide to the fifteenth antigen epitope peptide are shown in SEQ ID NOs. 1 to 15; the amino acid sequence of the sixteenth antigen epitope peptide is shown in SEQ ID NO. 16; the amino acid sequence of the seventeenth antigen epitope peptide is shown in SEQ ID NO. 1; the amino acid sequences of the eighteenth antigen epitope peptide to the twenty-first antigen epitope peptide are shown in SEQ ID NOs. 17 to 20; the amino acid sequence of the twenty-second antigen epitope peptide is shown in SEQ ID NO. 6; and the amino acid sequences of the twenty-third antigen epitope peptide to the twenty-sixth antigen epitope peptide are shown in SEQ ID NOs. 21 to 24.

[0006] As a preferred embodiment, the H1N1 antigen epitope peptide combination comprises a first antigen epitope peptide to a fifteenth antigen epitope peptide sequentially and discontinuously connected from the N-terminus to the C-terminus; the first antigen epitope peptide to the fifteenth antigen epitope peptide are connected via amino acids or polypeptides; From the N-terminus to the C-terminus, the H5N1 antigen epitope peptide combination includes the sixteenth antigen epitope peptide to the twenty-sixth antigen epitope peptide connected sequentially and discontinuously; the sixteenth antigen epitope peptide to the twenty-sixth antigen epitope peptide are connected through amino acids or polypeptides.

[0007] As a preferred embodiment, the amino acid sequence of the H1N1 antigen epitope peptide combination is shown as SEQ ID NO.25; the amino acid sequence of the H5N1 antigen epitope peptide combination is shown as SEQ ID NO.26.

[0008] The present invention also provides a gene composition, which includes a nucleotide sequence combination encoding the antigen epitope peptide combination described in the above scheme; the nucleotide sequence combination includes a nucleotide sequence encoding the H1N1 antigen epitope peptide combination and a nucleotide sequence encoding the H5N1 antigen epitope peptide combination.

[0009] As a preferred embodiment, the nucleotide sequence encoding the H1N1 antigen epitope peptide combination is shown as SEQ ID NO.27; the nucleotide sequence encoding the H5N1 antigen epitope peptide combination is shown as SEQ ID NO.28.

[0010] The present invention also provides the use of the antigen epitope peptide combination described in the above scheme or the gene composition described in the above scheme in the preparation of a drug for preventing influenza.

[0011] The present invention also provides the use of the antigen epitope peptide combination described in the above scheme or the gene composition described in the above scheme in the preparation of a broad-spectrum influenza vaccine.

[0012] The present invention also provides a broad-spectrum influenza vaccine, which comprises the antigen epitope peptide combination described in the above scheme.

[0013] As a preferred embodiment, the mass ratio of the H1N1 antigen epitope peptide combination to the H5N1 antigen epitope peptide combination is 1:1.

[0014] As a preferred embodiment, the broad-spectrum influenza vaccine further comprises an adjuvant.

[0015] Beneficial effects: The present invention provides an antigen epitope peptide combination for a broad-spectrum influenza vaccine, the antigen epitope peptide combination comprising an H1N1 antigen epitope peptide combination and an H5N1 antigen epitope peptide combination; the H1N1 antigen epitope peptide combination comprises a first antigen epitope peptide to a fifteenth antigen epitope peptide; the H5N1 antigen epitope peptide combination comprises a sixteenth antigen epitope peptide to a twenty-sixth antigen epitope peptide; the amino acid sequences of the first antigen epitope peptide to the fifteenth antigen epitope peptide are shown in SEQ ID NOs. 1 to 15; the amino acid sequence of the sixteenth antigen epitope peptide is shown in SEQ ID NO. 16; the amino acid sequence of the seventeenth antigen epitope peptide is shown in SEQ ID NO. 1; the amino acid sequences of the eighteenth antigen epitope peptide to the twenty-first antigen epitope peptide are shown in SEQ ID NOs. 17 to 20; the amino acid sequence of the twenty-second antigen epitope peptide is shown in SEQ ID NO. 6; and the amino acid sequences of the twenty-third antigen epitope peptide to the twenty-sixth antigen epitope peptide are shown in SEQ ID NOs. 21 to 24. The epitope peptide combination described in the present invention was screened from HA epitopes of H1N1 and H5N1 and can induce a broad-spectrum neutralizing antibody response in the host, allowing for the preparation of a broad-spectrum influenza vaccine. Using this epitope peptide combination, epitope fusion proteins Ag3 and Ag4 were screened and prepared into an Ag3+Ag4 influenza vaccine. Results from the examples showed that the Ag3+Ag4 influenza vaccine prepared in this invention reduced viral load in mice by 10-84 times compared to a blank control group and a trivalent influenza vaccine group. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0017] Figure 1 The three-dimensional structure of the H1N1 epitope fragment; the arrows in the figure indicate the beta-fold structure of the protein; Figure 2The three-dimensional structure of the H5N1 epitope fragment; Figure 3 The three-dimensional structures of Ag3 and Ag4 proteins predicted by AlphaFold software; A is the three-dimensional structure of Ag3 protein; B is the three-dimensional structure of Ag4 protein; the arrows in the figure indicate the beta-fold structure of the protein; Figure 4 The results of WB detection of Ag3 and Ag4 proteins; Figure 5 The results of SDS-PAGE electrophoresis of Ag3 and Ag4 proteins are shown; Figure 6 Figure 2 is the weight change of mice in each group; Figure 7 is the survival rate of mice in each group; Figure 8 These are the viral load graphs for each vaccine group; A is the viral load graph for A / Victoria / 2570 / 2019 (H1N1); B is the viral load graph for A / Darwin / 9 / 2021 (H3N2); and C is the viral load graph for B / Austria / 1359417 / 2021 (B / Victorialineage). DETAILED DESCRIPTION

[0018] The present invention provides an antigen epitope peptide combination for a broad-spectrum influenza vaccine, wherein the antigen epitope peptide combination comprises an H1N1 antigen epitope peptide combination and an H5N1 antigen epitope peptide combination; the H1N1 antigen epitope peptide combination comprises a first antigen epitope peptide to a fifteenth antigen epitope peptide; the H5N1 antigen epitope peptide combination comprises a sixteenth antigen epitope peptide to a twenty-sixth antigen epitope peptide; the amino acid sequences of the first antigen epitope peptide to the fifteenth antigen epitope peptide are shown in SEQ ID NOs. 1 to 15; the amino acid sequence of the sixteenth antigen epitope peptide is shown in SEQ ID NO. 16; the amino acid sequence of the seventeenth antigen epitope peptide is shown in SEQ ID NO. 1; the amino acid sequences of the eighteenth antigen epitope peptide to the twenty-first antigen epitope peptide are shown in SEQ ID NOs. 17 to 20; the amino acid sequence of the twenty-second antigen epitope peptide is shown in SEQ ID NO. 6; and the amino acid sequences of the twenty-third antigen epitope peptide to the twenty-sixth antigen epitope peptide are shown in SEQ ID NOs. 21 to 24. The H1N1 antigen epitope peptide combination of the present invention is a recognizable antigen epitope screened from 8 broad-spectrum influenza neutralizing antibodies, and the 8 broad-spectrum influenza neutralizing antibodies can neutralize influenza A Group 1 (H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17 and H18) and Group 2 (H3, H4, H7, H10, H14 and H15). The obtained H1N1 antigen epitope peptide combination can induce a targeted response of the above 8 broad-spectrum neutralizing antibodies, thereby achieving broad-spectrum immune protection against influenza A; the H5N1 antigen epitope peptide combination is a recognizable antigen epitope screened from 4 broad-spectrum influenza neutralizing antibodies; the 4 broad-spectrum influenza neutralizing antibodies can neutralize influenza A Group 1 and Group 2, as well as influenza B variants (B / Victoria lineage, B / Yamagata lineage), and the resulting H5N1 antigen epitope peptide combination can induce a targeted response of the above four influenza broad-spectrum neutralizing antibodies, thereby achieving a broad-spectrum immune response to influenza A and influenza B. The antigen epitope peptide combination of the present invention can induce the host to produce a broad-spectrum neutralizing antibody response and can be used to prepare a broad-spectrum influenza vaccine.

[0019] As an embodiment, from the N-terminus to the C-terminus, the H1N1 antigen epitope peptide combination includes the first antigen epitope peptide to the fifteenth antigen epitope peptide connected sequentially and discontinuously; the first antigen epitope peptide to the fifteenth antigen epitope peptide are connected by amino acids or polypeptides; in a specific embodiment of the present invention, the first antigen epitope peptide and the second antigen epitope peptide are connected by S; the second antigen epitope peptide and the third antigen epitope peptide are connected by DTSKKKNV (SEQ ID NO.29); the third antigen epitope peptide and the fourth antigen epitope peptide are connected by SLEELLKLGENKEQIELLLKEAKKKGV (SEQ ID NO.30); the fourth antigen epitope peptide and the fifth antigen epitope peptide are connected by TPLG (SEQ ID NO.31); the fifth antigen epitope peptide and the sixth antigen epitope peptide are connected by NVHELK (SEQ ID NO.32); the sixth antigen epitope peptide and the seventh antigen epitope peptide are connected by K; the seventh antigen epitope peptide and the eighth antigen epitope peptide are connected by PSLANNTEVGPGGLREVGGGKRSKDDP (SEQ ID NO.33); the eighth antigen epitope peptide and the ninth antigen epitope peptide are connected by W; the ninth antigen epitope peptide and the tenth antigen epitope peptide are connected by REYTAIGKTFNENEKEEEKKYKEEREKELKENTEYAENMV (SEQ ID NO.34); the tenth antigen epitope peptide and the eleventh antigen epitope peptide are connected by KIL; the eleventh antigen epitope peptide and the twelfth antigen epitope peptide are connected by RIEEL (SEQ ID NO.35); the twelfth antigen epitope peptide and the thirteenth antigen epitope peptide are connected by KQLKG (SEQ ID NO.36); the thirteenth antigen epitope peptide and the fourteenth antigen epitope peptide are connected by NG; the fourteenth antigen epitope peptide and the fifteenth antigen epitope peptide are connected by TFDYEK (SEQ ID NO.37); the N-terminus of the first antigen epitope peptide is further connected to YTLCV (SEQ ID NO.38).

[0020] In the present invention, from the N-terminus to the C-terminus, the H5N1 antigen epitope peptide combination includes the sixteenth antigen epitope peptide to the twenty-sixth antigen epitope peptide connected sequentially and discontinuously; the sixteenth antigen epitope peptide to the twenty-sixth antigen epitope peptide are connected through amino acids or polypeptides. In a specific embodiment of the present invention, the sixteenth antigen epitope peptide and the seventeenth antigen epitope peptide are connected by CV; the seventeenth antigen epitope peptide and the eighteenth antigen epitope peptide are connected by S; the eighteenth antigen epitope peptide and the nineteenth antigen epitope peptide are connected by DTPTEKNVT (SEQ ID NO.39); the nineteenth antigen epitope peptide and the twentieth antigen epitope peptide are connected by PEEIEKIKKLAKEETGKTVEVADC (SEQ ID NO.40); the twentieth antigen epitope peptide and the twenty-first antigen epitope peptide are connected by TILGGI (SEQ ID NO.41); the twenty-first antigen epitope peptide and the twenty-second antigen epitope peptide are connected by HPYY (SEQ ID NO.42); the twenty-second antigen epitope peptide and the twenty-third antigen epitope peptide are connected by K; the twenty-third antigen epitope peptide and the twenty-fourth antigen epitope peptide are connected by NFPASATSKPRPKLEYGFIGGIVPR (SEQ ID The twenty-fourth and twenty-fifth antigenic epitope peptides are linked via SGY; the twenty-fifth and twenty-sixth antigenic epitope peptides are linked via NSYKPEYKEFSEEEEEEKKKYKEEQEKKIEELTKEVEDTVLKTNQEILNDHDKYGKDLYQRVKDLLGDTAKDNGDGCFTFYA (SEQ ID NO. 44); the N-terminus of the sixteenth antigenic epitope peptide is further linked to A; the C-terminus of the twenty-sixth antigenic epitope peptide is further linked to TFDYEKYKEEAAKARAAELAG (SEQ ID NO. 45). The antigenic epitope peptides of the present invention are linked via amino acids or polypeptides, which can promote the reduction of the antigenic epitope peptide structure and induce the host to produce a broadly neutralizing antibody response.

[0021] In one embodiment, the amino acid sequence of the H1N1 antigen epitope peptide combination is shown in SEQ ID NO. 25, designated as Ag3 protein; and the amino acid sequence of the H5N1 antigen epitope peptide combination is shown in SEQ ID NO. 26, designated as Ag4 protein. The amino acid sequences of SEQ ID NO. 25 and SEQ ID NO. 26 of the present invention can improve the stability of Ag3 and Ag4 proteins and the degree of reduction of the antigenic epitope structure.

[0022] The present invention also provides a gene composition comprising a nucleotide sequence encoding the antigenic epitope peptide combination described in the above scheme; the nucleotide sequence combination comprises a nucleotide sequence encoding the H1N1 antigenic epitope peptide combination and a nucleotide sequence encoding the H5N1 antigenic epitope peptide combination. In one embodiment, the nucleotide sequence encoding the H1N1 antigenic epitope peptide combination is shown in SEQ ID NO. 27; the nucleotide sequence encoding the H5N1 antigenic epitope peptide combination is shown in SEQ ID NO. 28.

[0023] The present invention also provides the use of the antigen epitope peptide combination or the gene composition described in the above scheme in the preparation of a drug for preventing influenza.

[0024] The present invention also provides the use of the antigen epitope peptide combination or gene composition described in the above scheme in the preparation of a broad-spectrum influenza vaccine. The H1N1 antigen epitope peptide combination of the present invention can induce a targeted response of eight broadly neutralizing antibodies, achieving broad-spectrum immune protection against influenza A; the H5N1 antigen epitope peptide combination can induce a targeted response of four broadly neutralizing antibodies, achieving broad-spectrum immune protection against influenza A and influenza B. The combination of Ag3 and Ag4 antigens in the present invention can achieve targeted induction of a broadly neutralizing antibody response in the host, thereby further enhancing the vaccine's protective efficacy, achieving broad-spectrum protection against 18 influenza A strains (Group 1: H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, H18 and Group 2: H3, H4, H7, H10, H14, H15) and two influenza B variants (B / Victoria lineage and B / Yamagata lineage).

[0025] The present invention also provides a broad-spectrum influenza vaccine comprising the antigen epitope peptide combination described in the above scheme. In one embodiment, the mass ratio of the H1N1 antigen epitope peptide combination to the H5N1 antigen epitope peptide combination is 1:1.

[0026] In one embodiment, the broad-spectrum influenza vaccine further comprises an adjuvant. The present invention does not specifically limit the type of adjuvant; any vaccine adjuvant known to those skilled in the art can be used. In a specific embodiment of the present invention, the adjuvant is MF59. MF59 can be used as an adjuvant to reduce the antigen dosage and enhance the immune response.

[0027] To further illustrate the present invention, the antigen epitope peptide combination of a broad-spectrum influenza vaccine provided by the present invention and its application are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1: Acquisition of H1N1 epitope sequence and three-dimensional structure The antigenic epitopes recognized by broadly neutralizing antibodies play an important role in inducing the host to produce broadly neutralizing antibody responses. Therefore, eight H1N1 hemagglutinin (HA) antigen and broadly neutralizing antibody complex crystal structure files (PDB ID: 3ZTN, 8DIM, 7X6O, 5GJT, 7T3D, 7SCN, 7SCO, 8GV5) were downloaded from the Protein Data Bank (https: / / www.rcsb.org / ) database for the identification of important H1N1 antigenic epitopes. The 3ZTN corresponds to the broadly neutralizing antibody FI6, which can neutralize influenza variants Group 1 and Group 2. 2; the 8DIM corresponds to the broad-spectrum neutralizing antibody CR6261, which can neutralize influenza variants such as influenza A H1, H2, H5, H6, H8 and H9; the 7X6O corresponds to the broad-spectrum neutralizing antibody 28-12, which can neutralize influenza variants such as influenza A H1, H3, H4, and H7; the 5GJT corresponds to the broad-spectrum neutralizing antibody 3E1, which can neutralize influenza variants such as influenza A H1 and H5; the 7T3D corresponds to the broad-spectrum neutralizing antibody 22 2-1C06, which can neutralize influenza variants such as influenza A H1, H2, and H5; the 7SCN corresponds to the broad-spectrum neutralizing antibody 310-63E6, which can neutralize influenza variants such as influenza A H1 and H5; the 7SCO corresponds to the broad-spectrum neutralizing antibody 310-39G10, which can neutralize influenza variants such as influenza A H1 and H5; the 8GV5 corresponds to the broad-spectrum neutralizing antibody PN-SIA28, which can neutralize influenza variants Group A 1 and Group 2.

[0029] The antigenic epitopes recognized by the broad-spectrum neutralizing antibody in the complex of 8 H1N1 hemagglutinin antigen and broad-spectrum neutralizing antibody were obtained by IBEIP (V2.0) software. The epitope fragments of the 8 H1N1 antigen-antibody complexes were merged. After merging, all epitope fragments were extended by 2 amino acid residues to the amino terminus and the carboxyl terminus, respectively, as the H1N1 antigen epitope region, including the following 15 discontinuous epitope fragment sequences from the amino terminus to the carboxyl terminus: GYHAN (SEQ ID NO.1), STDTV (SEQ ID NO.2), TVTHSVNLLEDKHNGKLCKLRGV (SEQ ID NO.3), PVHDCDAKCQ (SEQ ID NO.4), AINSSLPFQ (SEQ ID NO.5), IGECPKYV (SEQ ID NO.6), STKLRLATGLRNI (SEQ ID NO.7), FIGG (SEQ ID NO.8), TGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMN (SEQ ID NO.9), LLENE (SEQ ID NO.10), DYHDS (SEQ ID NO.11), YEKVR (SEQ ID NO.12), NAKEIG (SEQ ID NO.13), CFEFYHKCDDECMESVKNG (SEQ ID NO.14), YSEEAKLNREEI (SEQ ID NO.15), which are recorded as the first to fifteenth antigen epitope peptides, respectively. The three-dimensional structure of the H1N1 epitope fragment is shown in FIG. Figure 1 As shown, the obtained H1N1 antigen epitope peptide can induce the targeted response of the above 8 broad-spectrum neutralizing antibodies, thereby achieving broad-spectrum immune protection against influenza A.

[0030] Example 2: Acquisition of H5N1 epitope sequence and three-dimensional structure To increase the number of broadly neutralizing antibody epitopes, four H5N1 hemagglutinin antigen-broadly neutralizing antibody complex crystal structure files (PDB IDs: 4FQI, 5JW4, 6E3H, and 3FKU) were downloaded from the Protein Data Bank (https: / / www.rcsb.org / ). 4FQI corresponds to the broadly neutralizing antibody CR9114, which neutralizes influenza A Group 1 and Group 2 strains, and influenza B strains B / Victoria lineage and B / Yamagata lineage; 5JW4 corresponds to the broadly neutralizing antibody MEDI8852, which neutralizes influenza A Group 1 and Group 2 strains; 6E3H corresponds to the broadly neutralizing antibody S9-3-37, which neutralizes influenza A Group 1 strain; and 3FKU corresponds to the broadly neutralizing antibody F10, which neutralizes influenza A H1, H2, H5, H6, H8, H9, and H11 strains.

[0031] The antigenic epitopes recognized by broad-spectrum neutralizing antibodies in the crystal structure files of four H5N1 hemagglutinin antigen-broad-spectrum neutralizing antibody complexes were obtained using IBEIP (V2.0) software. The epitope fragments of the four H5N1 antigen-antibody complexes were merged. After merging, all epitope fragments were extended by two amino acid residues to the amino terminus and the carboxyl terminus, respectively, as the H5N1 antigen epitope region, including the following 11 discontinuous epitope fragment sequences from the amino terminus to the carboxyl terminus: PGDQI (SEQ ID NO.16), GYHAN (SEQ ID NO.1), STEQV (SEQ ID NO.17), VTHAQDILEKKHNGKLCDLDGVKPL (SEQ ID NO.18), NTKCQ (SEQ ID NO.19), NSSMPFHNI (SEQ ID NO.20), IGECPKYV (SEQ ID NO.6), SNRLVLATGLR (SEQ ID NO.21), GWQGMVDGWYGYHHSNEQG (SEQ ID NO.22), and SVQVDGWYGYHHSNEQG (SEQ ID NO.23). NO.22), AADKESTQKAIDGVTNKVNSIIDKMN (SEQ ID NO.23), KCDNECMESVRNG (SEQ ID NO.24), which are recorded as the 16th to 26th antigen epitope peptides, respectively. The three-dimensional structure of the H5N1 epitope fragment is shown in FIG. Figure 2 As shown, the obtained H5N1 antigen epitope peptide can induce the targeted response of the above four broad-spectrum neutralizing antibodies, achieving broad-spectrum immune protection against influenza A and influenza B.

[0032] Example 3: Design of three-dimensional supporting protein structure of antigen epitope RFdiffusion software was used to design 5,000 protein support structures for discrete H1N1 and H5N1 epitope fragments. The protein support structures with the best structural stability (protein energy score) and conformational fidelity (Root Mean Square Deviation value) were selected. The obtained candidate protein support structures were then subjected to amino acid sequence design using ProteinMPNN software. The three-dimensional structures of the protein support structure sequences were predicted using AlphaFold software. The protein support structure sequences with the highest stability (H1N1 protein energy score: -499, H5N1 protein energy score: -625) and epitope structural fidelity (H1N1 RMSD: 0.63 Å, H5N1 RMSD: 0.65 Å) were selected.

[0033] The H1N1 epitope fragment and the supporting protein sequence (bold font in parentheses) are named Ag3 protein sequence (SEQ ID NO. 25): (YTLCV)GYHAN(S)STDTV(DTSKKKNV)TVTHSVNLLEDKHNGKLCKLRGV(SLEELLKLGENKEQIELLLKEAKKKGV)PVHDCDAKCQ(TPLG)AINSSLPFQ(NVHELK)IGECPKYV(K)STKLRLATGLRNI(PSLANNTEVGPGGLREVGGGKRSKDDP)FIEGG(W)TGMVD GWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMN(REYTAIGKTFNENEKEEEKKYKEEREKELKENTEYAENMV)LLENE(KIL)DYHDS(RIEEL)YEKVR(KQLKG)NAKEIG(NG)CFEFYHKCDDECMESVKNG(TFDYEK)YSEEAKLNREEI, AlphaFold software predicts the three-dimensional structure of Ag3 protein as follows Figure 3 As shown in A.

[0034] The H5N1 epitope fragment and the supporting protein sequence (bold font in parentheses) are named Ag4 protein sequence (SEQ IDNO.26): (A) PGDQI (CV) GYHAN (S) STEQV (DTPTEKNVT) VTHAQDILEKKHNGKLCDLDGVKPL (PEEIEKIKKLAKEETGKTVEVADC) NTKCQ (TILGGI) NSSMPFHNI (HPYY) IGECPKYV (K) SNRLVLATGLR (NFPASATSKPRPKLEYGFIGGIVPR) GWQGMVDGWYGYHHSNEQG (SGY) AADKESTQKAIDGVTNKVNSIIDKMN (NSYKPEYKEFSEEEEEEKKKYKEEQEKKIEELTKEVEDTVLKTNQEILNDHDKYGKDLYQRVKDLLGDTAKDNGDGCFTFYA) KCDNECMESVRNG (TFDYEKYKEEAAKARAAELAG), AlphaFold software predicted the three-dimensional structure of Ag4 protein as follows Figure 3 As shown in B.

[0035] Example 4: Preparation of Ag3 and Ag4 influenza vaccines Ag3 and Ag4 proteins were commissioned to Suzhou Hongxun Biotechnology Co., Ltd. for synthesis and expression. The gene sequences encoding the Ag3 protein and the Ag4 protein are shown in SEQ ID NO.27 and SEQ ID NO.28.

[0036]

[0037]

[0038] Plasmids expressing Ag3 and Ag4 proteins were synthesized and constructed respectively, and transfected into Expi293F cells. After 3 days of expression, the supernatant was centrifuged and purified. The proteins in the supernatant and cells were analyzed by Western Blot and SDS-PAGE electrophoresis. The proteins in the supernatant and cells were randomly divided into two groups. One group was marked as reduced, that is, reducing agents were added to the supernatant and cell proteins to destroy the three-dimensional structure of the protein, flattening the protein to form a linear structure; the other group was marked as non-reduced, that is, no reducing agent was added to keep the protein in its natural conformation. The test results are as follows: Figure 4 and Figure 5 shown.

[0039] according to Figure 4 and Figure 5 It can be seen that the molecular weight of Ag3 and Ag4 proteins is 40~55kd ( Figure 4 ), protein purity is greater than 90% ( Figure 5 ).

[0040] Example 5: Evaluation of the Immunity and Protection of Ag3 and Ag4 Broad-Spectrum Influenza Vaccines This study was conducted in 2023. To accurately evaluate the efficacy of the antigenic epitope peptide composition of the present invention as a vaccine, the trivalent influenza vaccine recommended by the World Health Organization (WHO) for the 2022-2023 pandemic strains was selected as a positive control. These included A / Victoria / 2570 / 2019 (H1N1), A / Darwin / 9 / 2021 (H3N2), and B / Austria / 1359417 / 2021 (B / Victoria lineage). It should be noted that this experiment only evaluated the efficacy of the antigenic epitope peptide composition of the present invention as a vaccine under the experimental conditions of this experiment, and does not affect its effectiveness in preventing and treating other viral strains in actual applications.

[0041] All animal experiments were approved by the Animal Experiment Ethics Committee of the Institute of Medical Biology, Chinese Academy of Medical Sciences and performed in strict accordance with the regulations of the Yunnan Provincial Laboratory Animal Welfare and Ethics Committee.

[0042] (1) Animal immunization BALB / c female mice, 6 to 8 weeks old, housed under a sterile environment were randomly divided into three groups of 5 to 6 mice each: a blank control group, an Ag3+Ag4 group, and a trivalent influenza vaccine group. Each group received three immunizations, two weeks apart, via intramuscular injection into the hind leg. The adjuvant was MF59 (Avitor).

[0043] Blank control group: 100 μL normal saline / mouse; Ag3+Ag4 group: Ag3 protein 10 μg / mouse, Ag4 protein 10 μg / mouse, MF59 adjuvant 50 μL / mouse; Trivalent influenza vaccine group: The trivalent influenza virus split vaccine (adult) recommended by the WHO for the 2022-2023 epidemic strains A / Victoria / 2570 / 2019 (H1N1), A / Darwin / 9 / 2021 (H3N2) and B / Austria / 1359417 / 2021 (B / Victoria lineage) was selected as the trivalent influenza vaccine group. The trivalent influenza virus split vaccine (adult) was purchased from Sanofi Pasteur Biological Products Co., Ltd. with batch number FL20220630, and 222 μL was injected into each mouse.

[0044] (2) Attack protection detection Step (1) Four weeks after the third immunization of each group of mice, the mice were challenged with nasal toxicity. The strains used in the challenge experiment were provided by the Institute of Medical Biology, Chinese Academy of Medical Sciences. The challenge dose was 50 μL 10 8 EID 50 A / Victoria / 2570 / 2019 (H1N1), 50μL 10 8 EID 50 A / Darwin / 9 / 2021 (H3N2), 50μL 10 8 EID 50 B / Austria / 1359417 / 2021 (B / Victoria lineage). The initial weight of the mice was recorded as 100%. Starting from the day of infection, the weight and survival rate of the mice were recorded every day. A weight loss of more than 25% was considered as death. The weight and survival rate of the mice in each group were recorded for 7 days. Figure 6-Figure 7 As shown in Tables 1 and 2.

[0045] Table 1 Body weight of mice in each group

[0046] Table 2 Survival rate of mice in each group

[0047] according to Figure 6-Figure 7 As shown in Tables 1 and 2, from 1 to 7 days after challenge, the body weight of mice in the Ag3+Ag4 group was 6% to 18.5% higher than that in the blank group ( Figure 6 ), the survival rate of mice in the Ag3+Ag4 group was 100%, while the survival rate of mice in the control group dropped to 75% from the 4th day and dropped to 0 on the 6th day ( Figure 7The body weight of mice in the Ag3+Ag4 group was 5.6% to 11.3% higher than that of the trivalent influenza vaccine group in the first 5 days after challenge, and was slightly lower than that of the trivalent influenza vaccine group in the last 2 days ( Figure 6 ), the survival rate of mice in the trivalent influenza vaccine group was the same as that in the Ag3+Ag4 group, both were 100%.

[0048] (3) Influenza viral load measurement Mice were sacrificed 7 days after challenge, and lung tissue was collected for RNA extraction and reverse transcription to obtain cDNA (Sevier, Cat. No. G3337). Three tubes were prepared for each reverse transcription product in a 0.1 mL PCR reaction plate. The reaction system consisted of 7.5 μL of 2× Universal Blue SYBRGreen qPCR Master Mix, 1.5 μL of 2.5 μM gene primers (upstream + downstream), 2.0 μL of reverse transcription product (cDNA), and 4.0 μL of Water Nuclease-Free.

[0049] Wuhan Sewell Biotechnology Co., Ltd. was commissioned to measure the viral load of A / Victoria / 2570 / 2019 (H1N1), A / Darwin / 9 / 2021 (H3N2), and B / Austria / 1359417 / 2021 (B / Victoria lineage) using qPCR. The qPCR amplification parameters were: 95°C for 30 seconds pre-denaturation; 95°C for 15 seconds denaturation; 60°C for 30 seconds annealing / extension for 40 cycles; 65°C → 95°C with fluorescence signal acquisition every 0.5°C increment. The qPCR extension primer sequences are shown in Table 3.

[0050] Table 3 qPCR primer sequence information

[0051] qPCR test results are as follows Figure 8 and as shown in Table 4.

[0052] Table 4 Viral load in each group

[0053] according to Figure 8As shown in Table 4, the viral loads of A / Victoria / 2570 / 2019 (H1N1) in the control group and the trivalent influenza vaccine group were 65 times and 10 times that of the Ag3+Ag4 group, respectively. The viral loads of A / Darwin / 9 / 2021 (H3N2) in the control group and the trivalent influenza vaccine group were 39.2 times and 12.7 times that of the Ag3+Ag4 group, respectively. The viral loads of B / Austria / 1359417 / 2021 (B / Victoria lineage) in the control group and the trivalent influenza vaccine group were 84 times and 18.4 times that of the Ag3+Ag4 group, respectively. As can be seen, the viral loads of the three viruses in the blank control group and the trivalent influenza vaccine group were 10 to 84 times that of the Ag3+Ag4 group, indicating that the viral load in the Ag3+Ag4 group was best controlled.

[0054] Ag3 and Ag4 proteins can be obtained by combining the antigenic epitope peptide combination of the broad-spectrum influenza vaccine of the present invention with a suitable supporting protein. As a vaccine, these proteins can induce multiple broad-spectrum neutralizing antibody responses and control the viral load at a low level.

[0055] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. An antigen epitope peptide combination for a broad-spectrum influenza vaccine, characterized in that: The antigen epitope peptide combination includes an H1N1 antigen epitope peptide combination and an H5N1 antigen epitope peptide combination; the H1N1 antigen epitope peptide combination includes the first antigen epitope peptide to the fifteenth antigen epitope peptide; the H5N1 antigen epitope peptide combination includes the sixteenth antigen epitope peptide to the twenty-sixth antigen epitope peptide; the amino acid sequences of the first antigen epitope peptide to the fifteenth antigen epitope peptide are shown in SEQ ID NO.1~15; the amino acid sequence of the sixteenth antigen epitope peptide is shown in SEQ ID NO.16; the amino acid sequence of the seventeenth antigen epitope peptide is shown in SEQ ID NO.1; the amino acid sequences of the eighteenth antigen epitope peptide to the twenty-first antigen epitope peptide are shown in SEQ ID NO.17~20; the amino acid sequence of the twenty-second antigen epitope peptide is shown in SEQ ID NO.6; the amino acid sequences of the twenty-third antigen epitope peptide to the twenty-sixth antigen epitope peptide are shown in SEQ ID NO.21~24.

2. The antigen epitope peptide combination according to claim 1, characterized in that: From the N-terminus to the C-terminus, the H1N1 antigen epitope peptide combination includes a first antigen epitope peptide to a fifteenth antigen epitope peptide that are sequentially and discontinuously connected; the first antigen epitope peptide to the fifteenth antigen epitope peptide are connected through amino acids or polypeptides; From the N-terminus to the C-terminus, the H5N1 antigen epitope peptide combination includes the sixteenth antigen epitope peptide to the twenty-sixth antigen epitope peptide which are sequentially and discontinuously connected; the sixteenth antigen epitope peptide to the twenty-sixth antigen epitope peptide are connected through amino acids or polypeptides.

3. The antigen epitope peptide combination according to claim 2, characterized in that: The amino acid sequence of the H1N1 antigen epitope peptide combination is shown in SEQ ID NO.25; the amino acid sequence of the H5N1 antigen epitope peptide combination is shown in SEQ ID NO.

26.

4. A gene composition, characterized in that: The gene composition comprises a nucleotide sequence combination encoding the antigen epitope peptide combination according to any one of claims 1 to 3; the nucleotide sequence combination comprises a nucleotide sequence encoding the H1N1 antigen epitope peptide combination and a nucleotide sequence encoding the H5N1 antigen epitope peptide combination.

5. The gene composition according to claim 4, characterized in that: The nucleotide sequence encoding the H1N1 antigen epitope peptide combination is shown in SEQ ID NO.27; the nucleotide sequence encoding the H5N1 antigen epitope peptide combination is shown in SEQ ID NO.

28.

6. Use of the antigen epitope peptide combination according to any one of claims 1 to 3 or the gene composition according to claim 4 or 5 in the preparation of a drug for preventing influenza.

7. Use of the antigen epitope peptide combination according to any one of claims 1 to 3 or the gene composition according to claim 4 or 5 in the preparation of a broad-spectrum influenza vaccine.

8. A broad-spectrum influenza vaccine, characterized in that: The broad-spectrum influenza vaccine comprises the antigen epitope peptide combination according to any one of claims 1 to 3.

9. The broad-spectrum influenza vaccine according to claim 8, characterized in that The mass ratio of the H1N1 antigen epitope peptide combination to the H5N1 antigen epitope peptide combination is 1:

1.

10. The broad-spectrum influenza vaccine according to claim 8 or 9, characterized in that: The broad-spectrum influenza vaccine also includes an adjuvant.

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