Antigenic epitope peptide combination of a broad-spectrum influenza vaccine and its application

Abstract

The present invention belongs to the field of biomedical technologies, and particularly relates to an antigenic epitope peptide combination of a broad-spectrum influenza vaccine and its application. The antigenic epitope peptide combination described in the present invention includes an H1N1 antigenic epitope peptide combination and an H5N1 antigenic epitope peptide combination. The H1N1 antigenic epitope peptide combination described in the present invention includes antigenic epitopes recognized by 8 broad-spectrum influenza neutralizing antibodies; the H5N1 antigenic epitope peptide combination includes antigenic epitopes recognized by another 4 broad-spectrum influenza neutralizing antibodies. Using the antigenic epitope peptide combination described in the present invention can induce a host to produce a broad-spectrum neutralizing antibody response and is used for preparing a broad-spectrum influenza vaccine. The results of the examples show that for the Ag3+Ag4 influenza vaccine prepared using the present invention, the viral load in mice decreased by 10 to 84 times compared with the blank control group and the trivalent influenza vaccine group.

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 every year. Although seasonal influenza vaccines are widely used for influenza prevention and control, existing vaccines often need to be updated every year to cope with different circulating virus strains due to continuous antigenic drift and transformation of the virus. This matching limitation has greatly affected the protective efficacy of seasonal influenza vaccines. Therefore, developing an influenza vaccine with broad-spectrum protection that can provide immune protection against multiple influenza strains without relying on specific virus strains has become a scientific problem that needs to be solved urgently.

[0003] As one of the most important structural proteins on the surface of influenza viruses, the hemagglutinin (HA) protein consists of a highly variable head and a relatively conservative stem. The neutralizing antibodies induced by certain antigenic epitope regions of the hemagglutinin (HA) protein can achieve a broad-spectrum neutralization effect independent of influenza variants, preventing the virus from invading host cells. However, the current immunization strategies targeting these conserved epitopes are still insufficient, 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 an important challenge in the field of influenza vaccine research and development. Summary of the invention

[0004] The purpose of the present invention is to provide an antigen epitope peptide combination for a broad-spectrum influenza vaccine and its application. The antigen 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 antigenic epitope peptide combination for a broad-spectrum influenza vaccine. The antigenic epitope peptide combination includes an H1N1 antigenic epitope peptide combination and an H5N1 antigenic epitope peptide combination. The H1N1 antigenic epitope peptide combination includes a first antigenic epitope peptide to a fifteenth antigenic epitope peptide. The H5N1 antigenic epitope peptide combination includes a sixteenth antigenic epitope peptide to a twenty-sixth antigenic epitope peptide. The amino acid sequences of the first antigenic epitope peptide to the fifteenth antigenic epitope peptide are as shown in SEQ ID NO.1 to 15. The amino acid sequence of the sixteenth antigenic epitope peptide is as shown in SEQ ID NO.16. The amino acid sequence of the seventeenth antigenic epitope peptide is as shown in SEQ ID NO.1. The amino acid sequences of the eighteenth antigenic epitope peptide to the twenty-first antigenic epitope peptide are as shown in SEQ ID NO.17 to 20. The amino acid sequence of the twenty-second antigenic epitope peptide is as shown in SEQ ID NO.6. The amino acid sequences of the twenty-third antigenic epitope peptide to the twenty-sixth antigenic epitope peptide are as shown in SEQ ID NO.21 to 24.

[0006] As a preferred embodiment, from the N-terminus to the C-terminus, the H1N1 antigenic epitope peptide combination includes the first antigenic epitope peptide to the fifteenth antigenic epitope peptide which are connected in sequence and discontinuously. The first antigenic epitope peptide to the fifteenth antigenic epitope peptide are connected by amino acids or polypeptides.

[0007] From the N-terminus to the C-terminus, the H5N1 antigenic epitope peptide combination includes the sixteenth antigenic epitope peptide to the twenty-sixth antigenic epitope peptide which are connected in sequence and discontinuously. The sixteenth antigenic epitope peptide to the twenty-sixth antigenic epitope peptide are connected by amino acids or polypeptides.

[0008] As a preferred embodiment, the amino acid sequence of the H1N1 antigenic epitope peptide combination is as shown in SEQ ID NO.25. The amino acid sequence of the H5N1 antigenic epitope peptide combination is as shown in SEQ ID NO.26.

[0009] The present invention also provides a gene composition. The gene composition includes a nucleotide sequence combination encoding the antigenic epitope peptide combination described in the above embodiment. The nucleotide sequence combination includes a nucleotide sequence encoding the H1N1 antigenic epitope peptide combination and a nucleotide sequence encoding the H5N1 antigenic epitope peptide combination.

[0010] As a preferred embodiment, the nucleotide sequence encoding the H1N1 antigenic epitope peptide combination is as shown in SEQ ID NO.27. The nucleotide sequence encoding the H5N1 antigenic epitope peptide combination is as shown in SEQ ID NO.28.

[0011] The present invention also provides the use of the antigenic epitope peptide combination described in the above embodiment or the gene composition described in the above embodiment in the preparation of a drug for preventing influenza.

[0012] The present invention also provides the use of the antigenic epitope peptide combination described in the above solution or the gene composition described in the above solution in the preparation of a broad-spectrum influenza vaccine.

[0013] The present invention also provides a broad-spectrum influenza vaccine, which includes the antigenic epitope peptide combination described in the above solution.

[0014] As a preferred solution, the mass ratio of the H1N1 antigenic epitope peptide combination to the H5N1 antigenic epitope peptide combination is 1:1.

[0015] As a preferred solution, the broad-spectrum influenza vaccine further includes an adjuvant.

[0016] Beneficial effects: The present invention provides an antigenic epitope peptide combination for a broad-spectrum influenza vaccine. The antigenic epitope peptide combination includes an H1N1 antigenic epitope peptide combination and an H5N1 antigenic epitope peptide combination; the H1N1 antigenic epitope peptide combination includes a first antigenic epitope peptide to a fifteenth antigenic epitope peptide; the H5N1 antigenic epitope peptide combination includes a sixteenth antigenic epitope peptide to a twenty-sixth antigenic epitope peptide; the amino acid sequences of the first antigenic epitope peptide to the fifteenth antigenic epitope peptide are as shown in SEQ ID NO.1 to 15; the amino acid sequence of the sixteenth antigenic epitope peptide is as shown in SEQ ID NO.16; the amino acid sequence of the seventeenth antigenic epitope peptide is as shown in SEQ ID NO.1; the amino acid sequences of the eighteenth antigenic epitope peptide to the twenty-first antigenic epitope peptide are as shown in SEQ ID NO.17 to 20; the amino acid sequence of the twenty-second antigenic epitope peptide is as shown in SEQ ID NO.6; the amino acid sequences of the twenty-third antigenic epitope peptide to the twenty-sixth antigenic epitope peptide are as shown in SEQ ID NO.21 to 24. The antigenic epitope peptide combination of the present invention is screened from the HA antigenic epitopes of H1N1 and H5N1, can induce a host to produce a broad-spectrum neutralizing antibody response, and is used for the preparation of a broad-spectrum influenza vaccine. Antigenic epitope fusion proteins Ag3 and Ag4 are screened using the antigenic epitope peptide combination of the present invention and prepared into an Ag3+Ag4 influenza vaccine. The results of the examples show that for the Ag3+Ag4 influenza vaccine prepared by the present invention, the viral load in mice is reduced by 10 to 84 times compared with the blank control group and the trivalent influenza vaccine group. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0018] Figure 1 It is a three-dimensional structure diagram of the H1N1 epitope fragment; the arrows in the figure indicate the beta-sheet structure of the protein;

[0019] Figure 2 It is the three-dimensional structure diagram of the H5N1 epitope fragment;

[0020] Figure 3 It is the three-dimensional structure diagram of the Ag3 protein and the Ag4 protein predicted by the AlphaFold software; where A is the three-dimensional structure of the Ag3 protein; B is the three-dimensional structure of the Ag4 protein; the arrows in the figure indicate the beta-sheet structure of the protein;

[0021] Figure 4 It is the WB detection result of the Ag3 and Ag4 proteins;

[0022] Figure 5 It is the SDS-PAGE electrophoresis detection result of the Ag3 and Ag4 proteins;

[0023] Figure 6 It is the graph of the body weight change of each group of mice;

[0024] Figure 7 It is the graph of the survival rate of each group of mice;

[0025] Figure 8 It is the graph of the viral load of each group of vaccines; where A is the graph of the viral load of A / Victoria / 2570 / 2019 (H1N1); B is the graph of the viral load of A / Darwin / 9 / 2021 (H3N2); C is the graph of the viral load of B / Austria / 1359417 / 2021 (B / Victorialineage). Specific implementation methods

[0026] The present invention provides an antigenic epitope peptide combination for a broad-spectrum influenza vaccine. The antigenic epitope peptide combination includes an H1N1 antigenic epitope peptide combination and an H5N1 antigenic epitope peptide combination. The H1N1 antigenic epitope peptide combination includes the first antigenic epitope peptide to the fifteenth antigenic epitope peptide. The H5N1 antigenic epitope peptide combination includes the sixteenth antigenic epitope peptide to the twenty-sixth antigenic epitope peptide. The amino acid sequences of the first antigenic epitope peptide to the fifteenth antigenic epitope peptide are as shown in SEQ ID NO.1 to 15. The amino acid sequence of the sixteenth antigenic epitope peptide is as shown in SEQ ID NO.16. The amino acid sequence of the seventeenth antigenic epitope peptide is as shown in SEQ ID NO.1. The amino acid sequences of the eighteenth antigenic epitope peptide to the twenty-first antigenic epitope peptide are as shown in SEQ ID NO.17 to 20. The amino acid sequence of the twenty-second antigenic epitope peptide is as shown in SEQ ID NO.6. The amino acid sequences of the twenty-third antigenic epitope peptide to the twenty-sixth antigenic epitope peptide are as shown in SEQ ID NO.21 to 24. The H1N1 antigenic epitope peptide combination of the present invention is an antigenic epitope screened from 8 broad-spectrum influenza neutralizing antibodies. 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 antigenic epitope peptide combination can induce a targeted response of the above 8 broad-spectrum neutralizing antibodies to achieve broad-spectrum immune protection against influenza A. The H5N1 antigenic epitope peptide combination is an antigenic 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 variant strains (B / Victoria lineage, B / Yamagata lineage). The obtained H5N1 antigenic epitope peptide combination can induce a targeted response of the above 4 broad-spectrum influenza neutralizing antibodies to achieve broad-spectrum immune responses against influenza A and influenza B. Using the antigenic epitope peptide combination of the present invention can induce a host to produce a broad-spectrum neutralizing antibody response and can be used for preparing a broad-spectrum influenza vaccine.

[0027] As an embodiment, from the N-terminus to the C-terminus, the H1N1 antigenic epitope peptide combination comprises the first to the fifteenth antigenic epitope peptides that are sequentially and discontinuously linked; the first to the fifteenth antigenic epitope peptides are linked by amino acids or polypeptides; in a specific embodiment of the present invention, the first antigenic epitope peptide and the second antigenic epitope peptide are linked by S; the second antigenic epitope peptide and the third antigenic epitope peptide are linked by DTSKKKNV (SEQ ID NO.29); the third antigenic epitope peptide and the fourth antigenic epitope peptide are linked by SLEELLKLGENKEQIELLLKEAKKKGV (SEQ ID NO.30); the fourth antigenic epitope peptide and the fifth antigenic epitope peptide are linked by TPLG (SEQ IDNO.31); the fifth antigenic epitope peptide and the sixth antigenic epitope peptide are linked by NVHELK (SEQ ID NO.32); the sixth antigenic epitope peptide and the seventh antigenic epitope peptide are linked by K; the seventh antigenic epitope peptide and the eighth antigenic epitope peptide are linked by PSLANNTEVGPGGLREVGGGKRSKDDP (SEQ ID NO.33); the eighth antigenic epitope peptide and the ninth antigenic epitope peptide are linked by W; the ninth antigenic epitope peptide and the tenth antigenic epitope peptide are linked by REYTAIGKTFNENEKEEEKKYKEEREKELKENTEYAENMV (SEQ ID NO.34); the tenth antigenic epitope peptide and the eleventh antigenic epitope peptide are linked by KIL; the eleventh antigenic epitope peptide and the twelfth antigenic epitope peptide are linked by RIEEL (SEQ ID NO.35); the twelfth antigenic epitope peptide and the thirteenth antigenic epitope peptide are linked by KQLKG (SEQ ID NO.36); the thirteenth antigenic epitope peptide and the fourteenth antigenic epitope peptide are linked by NG; the fourteenth antigenic epitope peptide and the fifteenth antigenic epitope peptide are linked by TFDYEK (SEQ ID NO.37); the N-terminus of the first antigenic epitope peptide is further linked to YTLCV (SEQ ID NO.38).

[0028] In the present invention, from the N-terminus to the C-terminus, the H5N1 antigenic epitope peptide combination includes the sixteenth antigenic epitope peptide to the twenty-sixth antigenic epitope peptide that are sequentially and discontinuously connected; the sixteenth antigenic epitope peptide to the twenty-sixth antigenic epitope peptide are connected by amino acids or polypeptides. In a specific embodiment of the present invention, the sixteenth antigenic epitope peptide and the seventeenth antigenic epitope peptide are connected by CV; the seventeenth antigenic epitope peptide and the eighteenth antigenic epitope peptide are connected by S; the eighteenth antigenic epitope peptide and the nineteenth antigenic epitope peptide are connected by DTPTEKNVT (SEQ ID NO.39); the nineteenth antigenic epitope peptide and the twentieth antigenic epitope peptide are connected by PEEIEKIKKLAKEETGKTVEVADC (SEQ ID NO.40); the twentieth antigenic epitope peptide and the twenty-first antigenic epitope peptide are connected by TILGGI (SEQ ID NO.41); the twenty-first antigenic epitope peptide and the twenty-second antigenic epitope peptide are connected by HPYY (SEQ ID NO.42); the twenty-second antigenic epitope peptide and the twenty-third antigenic epitope peptide are connected by K; the twenty-third antigenic epitope peptide and the twenty-fourth antigenic epitope peptide are connected by NFPASATSKPRPKLEYGFIGGIVPR (SEQ ID NO.43); the twenty-fourth antigenic epitope peptide and the twenty-fifth antigenic epitope peptide are connected by SGY; the twenty-fifth antigenic epitope peptide and the twenty-sixth antigenic epitope peptide are connected by NSYKPEYKEFSEEEEEEKKKYKEEQEKKIEELTKEVEDTVLKTNQEILNDHDKYGKDLYQRVKDLLGDTAKDNGDGCFTFYA (SEQ ID NO.44); A is further connected to the N-terminus of the sixteenth antigenic epitope peptide; TFDYEKYKEEAAKARAAELAG (SEQ ID NO.45) is further connected to the C-terminus of the twenty-sixth antigenic epitope peptide. The antigenic epitope peptides of the present invention are connected by amino acids or polypeptides, which can promote the reduction of the antigenic epitope peptide structure and induce the host to produce a broad-spectrum neutralizing antibody response.

[0029] As an embodiment, the amino acid sequence of the H1N1 antigenic epitope peptide combination is as shown in SEQ ID NO.25, named Ag3 protein; the amino acid sequence of the H5N1 antigenic epitope peptide combination is as shown in SEQ ID NO.26, named Ag4 protein. The amino acid sequences shown in SEQ ID NO.25 and SEQ ID NO.26 of the present invention can improve the stability of Ag3 protein and Ag4 protein and the reduction degree of the antigenic epitope structure.

[0030] The present invention also provides a gene composition, which comprises a nucleotide sequence combination encoding the antigenic epitope peptide combination described in the above solution; 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. As an embodiment, the nucleotide sequence encoding the H1N1 antigenic epitope peptide combination is as shown in SEQ ID NO.27; the nucleotide sequence encoding the H5N1 antigenic epitope peptide combination is as shown in SEQ ID NO.28.

[0031] The present invention also provides the use of the antigenic epitope peptide combination or the gene composition described in the above solution in the preparation of a drug for preventing influenza.

[0032] The present invention also provides the use of the antigenic epitope peptide combination or the gene composition described in the above solution in the preparation of a broad-spectrum influenza vaccine. The H1N1 antigenic epitope peptide combination of the present invention can induce a targeted response of 8 broad-spectrum neutralizing antibodies to achieve broad-spectrum immune protection against influenza A; the H5N1 antigenic epitope peptide combination can induce a targeted response of 4 broad-spectrum neutralizing antibodies to achieve broad-spectrum immune protection against influenza A and influenza B. Through the combination of Ag3 and Ag4 antigens, the present invention can achieve targeted induction of a broad-spectrum influenza neutralizing antibody response in the host, thereby further enhancing the protection efficiency of the vaccine and achieving broad-spectrum protection against 18 influenza A viruses (Influenza A Group 1: H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, H18 and Group 2: H3, H4, H7, H10, H14, H15) and 2 influenza B virus variants (B / Victoria lineage, B / Yamagata lineage).

[0033] The present invention also provides a broad-spectrum influenza vaccine, which comprises the antigenic epitope peptide combination described in the above solution. As an embodiment, the mass ratio of the H1N1 antigenic epitope peptide combination to the H5N1 antigenic epitope peptide combination is 1:1.

[0034] As an embodiment, the broad-spectrum influenza vaccine further comprises an adjuvant. The present invention has no special limitation on the type of the adjuvant, and the types of vaccine adjuvants well-known to those skilled in the art can be used. In a specific embodiment of the present invention, the adjuvant is MF59, and MF59 as an adjuvant can reduce the antigen dosage and enhance the intensity of the immune response.

[0035] In order to further illustrate the present invention, the antigenic epitope peptide combination of a broad-spectrum influenza vaccine provided by the present invention and its application will be described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0036] Example 1: Obtaining H1N1 Epitope Sequences and Three-Dimensional Structures

[0037] Antigenic epitopes recognized by broadly neutralizing antibodies play an important role in inducing a broadly neutralizing antibody response in the host. Therefore, crystal structure files of 8 H1N1 hemagglutinin (HA) antigens complexed with broadly neutralizing antibodies were downloaded from the Protein Data Bank (https: / / www.rcsb.org / ) database (PDB IDs: 3ZTN, 8DIM, 7X6O, 5GJT, 7T3D, 7SCN, 7SCO, 8GV5) for the identification of important H1N1 antigenic epitopes. The 3ZTN corresponds to the broadly neutralizing antibody FI6, which can neutralize influenza variants of influenza A groups 1 and 2; the 8DIM corresponds to the broadly neutralizing antibody CR6261, which can neutralize influenza variants of influenza A H1, H2, H5, H6, H8, and H9; the 7X6O corresponds to the broadly neutralizing antibody 28-12, which can neutralize influenza variants of influenza A H1, H3, H4, and H7; the 5GJT corresponds to the broadly neutralizing antibody 3E1, which can neutralize influenza variants of influenza A H1 and H5; the 7T3D corresponds to the broadly neutralizing antibody 222-1C06, which can neutralize influenza variants of influenza A H1, H2, and H5; the 7SCN corresponds to the broadly neutralizing antibody 310-63E6, which can neutralize influenza variants of influenza A H1 and H5; the 7SCO corresponds to the broadly neutralizing antibody 310-39G10, which can neutralize influenza variants of influenza A H1 and H5; the 8GV5 corresponds to the broadly neutralizing antibody PN-SIA28, which can neutralize influenza variants of influenza A groups 1 and 2.

[0038] The antigenic epitopes recognized by the broad-spectrum neutralizing antibodies in 8 H1N1 hemagglutinin antigen-broad-spectrum neutralizing antibody complexes were obtained by the IBEIP (V2.0) software. The epitope fragments of the 8 H1N1 antigen-antibody complexes were combined. After combination, all epitope fragments were extended by 2 amino acid residues towards the amino terminus and the carboxyl terminus respectively, serving as the H1N1 antigenic 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), FIEGG (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 were sequentially denoted as the first antigenic epitope peptide to the fifteenth antigenic epitope peptide. The three-dimensional structure of the H1N1 epitope fragment is as Figure 1 shown, and the obtained H1N1 antigenic epitope peptides can induce the targeted responses of the above 8 broad-spectrum neutralizing antibodies, achieving broad-spectrum immune protection against influenza A.

[0039] Example 2: Obtaining the H5N1 epitope sequence and three-dimensional structure

[0040] To increase the epitopes of broad-spectrum neutralizing antibodies, four crystal structure files of H5N1 hemagglutinin antigen and broad-spectrum neutralizing antibody complexes (PDB IDs: 4FQI, 5JW4, 6E3H, 3FKU) were downloaded from the Protein Data Bank (https: / / www.rcsb.org / ) database. The 4FQI corresponds to the broad-spectrum neutralizing antibody CR9114, which can neutralize influenza variants including influenza A Group 1, Group 2, influenza B variants B / Victoria lineage and B / Yamagata lineage; the 5JW4 corresponds to the broad-spectrum neutralizing antibody MEDI8852, which can neutralize influenza variants of influenza A Group 1 and Group 2; the 6E3H corresponds to the broad-spectrum neutralizing antibody S9-3-37, which can neutralize influenza variants of influenza A Group 1; the 3FKU corresponds to the broad-spectrum neutralizing antibody F10, which can neutralize influenza variants of influenza A H1, H2, H5, H6, H8, H9, H11.

[0041] The antigen epitopes recognized by the broad-spectrum neutralizing antibodies in the four crystal structure files of H5N1 hemagglutinin antigen and broad-spectrum neutralizing antibody complexes were obtained by IBEIP (V2.0) software. The epitope fragments of the four H5N1 antigen-antibody complexes were combined. After combination, all epitope fragments were extended by 2 amino acid residues towards the amino terminus and carboxyl terminus respectively to serve 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 IDNO.19), NSSMPFHNI (SEQ ID NO.20), IGECPKYV (SEQ ID NO.6), SNRLVLATGLR (SEQ IDNO.21), GWQGMVDGWYGYHHSNEQG (SEQ ID NO.22), AADKESTQKAIDGVTNKVNSIIDKMN (SEQ IDNO.23), KCDNECMESVRNG (SEQ ID NO.24), which were sequentially denoted as the sixteenth antigen epitope peptide to the twenty-sixth antigen epitope peptide. The three-dimensional structure of the H5N1 epitope fragment is as Figure 2 shown, and the obtained H5N1 antigen epitope peptides can induce the targeted responses of the above 4 broad-spectrum neutralizing antibodies, achieving broad-spectrum immune protection against influenza A and influenza B.

[0042] Example 3: Three-dimensional supporting protein structure design of antigen epitopes

[0043] 5,000 protein support structures were designed for discontinuous H1N1 and H5N1 antigenic epitope fragments by the RFdiffusion software, and the protein support structures with the optimal structural stability (protein energy score) and conformational restoration degree (Root Mean Square Deviation value) were selected. The amino acid sequences of the obtained candidate support protein structures were designed by the ProteinMPNN software. The three-dimensional structures of the protein support structure sequences were predicted by the AlphaFold software, and the protein support structure sequences with the highest stability (H1N1 protein energy score: -499, H5N1 protein energy score: -625) and antigenic epitope structure restoration degree (H1N1 RMSD: 0.63 Å, H5N1 RMSD: 0.65 Å) were selected.

[0044] The H1N1 epitope fragment and the support protein sequence (bold font in parentheses) were named the Ag3 protein sequence (SEQ ID NO.25): (YTLCV)GYHAN(S)STDTV(DTSKKKNV)TVTHSVNLLEDKHNGKLCKLRGV(SLEELLKLGENKEQIELLLKEAKKKGV)PVHDCDAKCQ(TPLG)AINSSLPFQ(NVHELK)IGECPKYV(K)STKLRLATGLRNI(PSLANNTEVGPGGLREVGGGKRSKDDP)FIEGG(W)TGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMN(REYTAIGKTFNENEKEEEKKYKEEREKELKENTEYAENMV)LLENE(KIL)DYHDS(RIEEL)YEKVR(KQLKG)NAKEIG(NG)CFEFYHKCDDECMESVKNG(TFDYEK)YSEEAKLNREEI. The three-dimensional structure of the Ag3 protein predicted by the AlphaFold software is as shown in Figure 3 A in

[0045] The H5N1 epitope fragment and the supporting protein sequence (bold font in parentheses) are named the Ag4 protein sequence (SEQ ID NO.26): (A)PGDQI(CV)GYHAN(S)STEQV(DTPTEKNVT)VTHAQDILEKKHNGKLCDLDGVKPL(PEEIEKIKKLAKEETGKTVEVADC)NTKCQ(TILGGI)NSSMPFHNI(HPYY)IGECPKYV(K)SNRLVLATGLR(NFPASATSKPRPKLEYGFIGGIVPR)GWQGMVDGWYGYHHSNEQG(SGY)AADKESTQKAIDGVTNKVNSIIDKMN(NSYKPEYKEFSEEEEEEKKKYKEEQEKKIEELTKEVEDTVLKTNQEILNDHDKYGKDLYQRVKDLLGDTAKDNGDGCFTFYA)KCDNECMESVRNG(TFDYEKYKEEAAKARAAELAG). The three-dimensional structure of the Ag4 protein predicted by the AlphaFold software is as shown in B of Figure 3 shown below.

[0046] Example 4: Preparation of Ag3 and Ag4 influenza vaccines

[0047] The Ag3 and Ag4 proteins were commissioned to be synthesized and expressed by Suzhou Hongxun Biotechnology Co., Ltd. The gene sequences encoding the Ag3 protein and the Ag4 protein are shown in SEQ ID NO.27 and SEQ ID NO.28.

[0048]

[0049]

[0050] Plasmids expressing Ag3 and Ag4 proteins were synthesized and constructed separately, transfected into Expi293F cells. After 3 days of expression, the cells were centrifuged and the supernatant was taken for protein purification. Western Blot was used to detect and SDS-PAGE electrophoresis was used to analyze the proteins in the supernatant and cells. The proteins in the supernatant and cells were randomly divided into two groups. One group was recorded as the reduced group, that is, a reducing agent was added to the proteins in the supernatant and cells respectively to destroy the three-dimensional structure of the proteins and make the proteins flatten into a linear structure; the other group was recorded as the non-reduced group, that is, no reducing agent was added to keep the proteins in their native conformation. The detection results are as Figure 4 and Figure 5 shown.

[0051] According to Figure 4 and Figure 5 it can be known that the molecular weights of Ag3 and Ag4 proteins are 40 - 55 kd ( Figure 4 ), and the protein purity is greater than 90% ( Figure 5 ).

[0052] Example 5: Immune and protective evaluation of Ag3 and Ag4 broad-spectrum influenza vaccines

[0053] This study was carried out in 2023. In order to accurately evaluate the effect of the antigenic epitope peptide composition involved in the present invention as a vaccine, trivalent influenza vaccines recommended by the World Health Organization (WHO) in 2022 - 2023 for the current epidemic strains were selected as positive controls, including A / Victoria / 2570 / 2019 (H1N1), A / Darwin / 9 / 2021 (H3N2), and B / Austria / 1359417 / 2021 (B / Victoria lineage). It should be particularly noted that this experiment only evaluated the effect of the antigenic epitope peptide composition of the present invention as a vaccine for these three specific epidemic strains under the experimental conditions, and does not affect its prevention and treatment effects on other virus strains in actual applications.

[0054] All animal experiments were approved by the Animal Experiment Ethics Committee of the Institute of Medical Biology, Chinese Academy of Medical Sciences, and were strictly operated in accordance with the regulations of the Yunnan Laboratory Animal Welfare and Ethics Committee.

[0055] (1) Animal immunization

[0056] Female BALB / c mice aged 6 - 8 weeks raised in a sterile environment were randomly divided into 3 groups, with 5 to 6 mice in each group, and were successively recorded as the blank control group, the Ag3 + Ag4 group, and the trivalent influenza vaccine group. Each group of mice was immunized three times, with a two-week interval between two immunizations. The immunization was performed by intramuscular injection in the hind legs, and the immune adjuvant was MF59 (Aivitop).

[0057] Blank control group: 100 μL of normal saline per mouse;

[0058] Ag3 + Ag4 group: 10 μg of Ag3 protein per mouse, 10 μg of Ag4 protein per mouse, and 50 μL of MF59 adjuvant per mouse;

[0059] Trivalent influenza vaccine group: The trivalent influenza virus split vaccine (for adults) recommended by the WHO for the current epidemic strains A / Victoria / 2570 / 2019 (H1N1), A / Darwin / 9 / 2021 (H3N2), and B / Austria / 1359417 / 2021 (B / Victoria lineage) from 2022 to 2023 was used as the trivalent influenza vaccine group. The trivalent influenza virus split vaccine (for adults) was purchased from Sanofi Pasteur Biologics Co., Ltd., with the batch number FL20220630, and 222 μL was injected into each mouse.

[0060] (2) Viral challenge protection assay

[0061] Four weeks after the third immunization of the mice in each group in step (1), the mice were challenged nasally. The viral strains used in the challenge experiment were all provided by the Institute of Medical Biology, Chinese Academy of Medical Sciences. The challenge dose was 50 μL of 10 8 EID 50 A / Victoria / 2570 / 2019 (H1N1), 50 μL of 10 8 EID 50 A / Darwin / 9 / 2021 (H3N2), 50 μL of 10 8 EID 50 B / Austria / 1359417 / 2021 (B / Victoria lineage). The initial body weight of the mice was recorded as 100%. Starting from the day of challenge, the body weight and survival rate of the mice were recorded every day. A weight loss of more than 25% was considered the death of the mouse. The recording was carried out for 7 days. The body weight and survival rate of the mice in each group are as shown in Figures 6 - 7 Tables 1 and 2.

[0062] Table 1 Body weight of mice in each group

[0063]

[0064] Table 2 Survival rate of mice in each group

[0065]

[0066] According to Figures 6 - 7 Tables 1 and 2, from 1 to 7 days after the challenge, the body weight of the mice in the Ag3 + Ag4 group was 6% to 18.5% higher than that of the blank group ( Figure 6), the survival rates of the mice in the Ag3+Ag4 group were both 100%. The survival rate of the control group mice decreased to 75% starting from the 4th day and dropped to 0 on the 6th day ( Figure 7 ). In the first 5 days after virus challenge, the body weights of the mice in the Ag3+Ag4 group were 5.6% to 11.3% higher than those in the trivalent influenza vaccine group, and slightly lower than those in the trivalent influenza vaccine group in the following 2 days ( Figure 6 ). The survival rate of the mice in the trivalent influenza vaccine group was the same as that in the Ag3+Ag4 group, both being 100%.

[0067] (3) Determination of influenza virus load

[0068] The mice were sacrificed 7 days after virus challenge, and lung tissues were taken to extract RNA, and cDNA was obtained by reverse transcription (Saiweier, product number: G3337). Take a 0.1 mL PCR reaction plate, and prepare 3 tubes for each reverse transcription product. The reaction system is: 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.

[0069] Wuhan Saiweier Biotechnology Co., Ltd. was commissioned to use qPCR to determine the virus loads of A / Victoria / 2570 / 2019 (H1N1), A / Darwin / 9 / 2021 (H3N2), and B / Austria / 1359417 / 2021 (B / Victoria lineage). The qPCR amplification parameters are: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 15 s, annealing / extension at 60°C for 30 s, 40 cycles; from 65°C to 95°C, and fluorescence signals are collected every 0.5°C increase in temperature. The qPCR amplification primer sequences are shown in Table 3.

[0070] Table 3 qPCR primer sequence information

[0071]

[0072] The qPCR detection results are as Figure 8 and Table 4 show.

[0073] Table 4 Virus loads of each group

[0074]

[0075] According to Figure 8As can be seen from 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. It can be seen that 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. Therefore, the viral load of the Ag3+Ag4 group was the best controlled.

[0076] The antigenic epitope peptide combination of the broad-spectrum influenza vaccine described in the present invention can be combined with a suitable supporting protein to obtain Ag3 and Ag4 proteins. As a vaccine, it can induce a multiple broad-spectrum neutralizing antibody response and can control the viral load at a low level.

[0077] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments according to these embodiments without creative efforts, and these embodiments all belong to the protection scope of the present invention.

Claims

1. An antigenic epitope peptide combination for a broad-spectrum influenza vaccine, characterized in that, The antigenic epitope peptide combination includes an H1N1 antigenic epitope peptide combination and an H5N1 antigenic epitope peptide combination; the H1N1 antigenic epitope peptide combination includes the first to the fifteenth antigenic epitope peptides; the H5N1 antigenic epitope peptide combination includes the sixteenth to the twenty-sixth antigenic epitope peptides; the amino acid sequences of the first to the fifteenth antigenic epitope peptides are as shown in SEQ ID NO.1~15; the amino acid sequence of the sixteenth antigenic epitope peptide is as shown in SEQ ID NO.16; the amino acid sequence of the seventeenth antigenic epitope peptide is as shown in SEQ ID NO.1; the amino acid sequences of the eighteenth to the twenty-first antigenic epitope peptides are as shown in SEQ ID NO.17~20; the amino acid sequence of the twenty-second antigenic epitope peptide is as shown in SEQ ID NO.6; the amino acid sequences of the twenty-third to the twenty-sixth antigenic epitope peptides are as shown in SEQ ID NO.21~24; From the N-terminus to the C-terminus, the H1N1 antigenic epitope peptide combination includes the first to the fifteenth antigenic epitope peptides that are sequentially and discontinuously linked; the first to the fifteenth antigenic epitope peptides are linked by amino acids or polypeptides; From the N-terminus to the C-terminus, the H5N1 antigenic epitope peptide combination includes the sixteenth to the twenty-sixth antigenic epitope peptides that are sequentially and discontinuously linked; the sixteenth to the twenty-sixth antigenic epitope peptides are linked by amino acids or polypeptides.

2. The antigenic epitope peptide combination according to claim 1, characterized in that, The amino acid sequence of the H1N1 antigenic epitope peptide combination is as shown in SEQ ID NO.25; the amino acid sequence of the H5N1 antigenic epitope peptide combination is as shown in SEQ ID NO.

26.

3. A gene composition, characterized in that, The gene composition includes a nucleotide sequence combination encoding the antigenic epitope peptide combination according to claim 1 or 2; the nucleotide sequence combination includes a nucleotide sequence encoding the H1N1 antigenic epitope peptide combination and a nucleotide sequence encoding the H5N1 antigenic epitope peptide combination.

4. The gene composition according to claim 3, wherein The nucleotide sequence encoding the H1N1 antigenic epitope peptide combination is as shown in SEQ ID NO.27; the nucleotide sequence encoding the H5N1 antigenic epitope peptide combination is as shown in SEQ ID NO.

28.

5. Use of the antigenic epitope peptide combination according to claim 1 or 2 or the gene composition according to claim 3 or 4 in the preparation of a drug for preventing influenza.

6. Use of the antigenic epitope peptide combination according to claim 1 or 2 or the gene composition according to claim 3 or 4 in the preparation of a broad-spectrum influenza vaccine.

7. A broad-spectrum influenza vaccine, characterized in that, The broad-spectrum influenza vaccine includes the antigenic epitope peptide combination according to claim 1 or 2.

8. The broad-spectrum influenza vaccine according to claim 7, wherein The mass ratio of the H1N1 antigenic epitope peptide combination to the H5N1 antigenic epitope peptide combination is 1:

1.

9. The broad-spectrum influenza vaccine according to claim 7 or 8, characterized in that, The broad-spectrum influenza vaccine further includes an adjuvant.

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