Multi-epitope peptide composite vaccine based on self-assembly principle as well as preparation method and application of multi-epitope peptide composite vaccine

Abstract

The invention relates to a multi-epitope peptide composite vaccine based on a self-assembly principle as well as a preparation method and application of the multi-epitope peptide composite vaccine. The multi-epitope peptide composite vaccine comprises an antigen epitope peptide of a virus and a nucleic acid adjuvant, and the antigen epitope peptide of the virus and the nucleic acid adjuvant are self-assembled through electrostatic attraction to form a nano-composite without a periodic microstructure or a nano-crystal with a periodic microstructure. The multi-epitope peptide composite vaccine takes polypeptide as a specific antigen and is used for activating a specific immune response of an organism; the nucleic acid adjuvant is used for activating the innate immune system of the body and enhancing the immunogenicity of the epitope polypeptide. According to the scheme, the nanocrystal with the periodic microstructure can obviously induce recruitment and activation of a nucleic acid receptor of a host cell; various epitope peptides can be integrated into the same nanocrystal, so that the scheme simultaneously improves the immunogenicity and broad spectrum of a polypeptide vaccine.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a multi-epitope peptide composite vaccine based on the self-assembly principle and a preparation method and application thereof. Background Art

[0002] An epitope peptide is a short peptide that can be recognized and bound by the immune system, usually derived from the antigenic protein of a pathogen. It can bind to the human immunocompatibility complex (MHC) and be presented to T cells or B cells, thereby forming an antigen-specific immune response. By directly preparing epitope peptides into vaccines, it is theoretically possible to more effectively train the body to recognize and resist pathogenic microorganisms. However, the design of epitope peptide vaccines faces multiple challenges. First, accurate identification and selection of effective epitopes are crucial, but this process is extremely difficult due to the diversity of pathogens and the complexity of epitope structures. Secondly, epitope peptides have low immunogenicity and often need to be combined with suitable adjuvants to enhance the immune response, which further increases the complexity of vaccine design. In addition, epitope peptides are easily degraded by the body, and how to improve their stability is also an important difficulty in design.

[0003] Influenza virus is the main pathogen of seasonal influenza outbreaks, infecting about one billion people worldwide each year, of which 3 to 5 million develop severe symptoms. During the flu season, there are multiple influenza variants coexisting. As of the end of 2023, test data showed that 79.1% of the strains belonged to influenza A virus, of which 72.9% were H1N1 subtypes and 27.1% were H3N2 subtypes. Among influenza B viruses, most belong to the Victoria lineage, accounting for 21.5% of the presidential count. At present, influenza vaccines are mainly used to reduce the spread of influenza viruses and reduce the mortality rate of patients.

[0004] Currently available influenza vaccines generally activate the body's immune system through inactivated influenza viruses or recombinant influenza proteins, enabling it to quickly activate and neutralize and eliminate the virus after influenza virus infection. Hemagglutinin (HA) is one of the surface proteins that enables influenza viruses to adhere to the host cell membrane and induce the formation of fusion pores to achieve viral entry. Therefore, HA protein is usually used as the main target for vaccine design. The World Health Organization publishes recommended strains for influenza vaccines every year based on epidemiological surveys of influenza worldwide.

[0005] However, the epitope information of HA protein may vary significantly among influenza epidemic strains, and the resulting antigenic drift or shift may significantly reduce the immune efficacy of the vaccine. Although various technologies have been used to improve the accuracy of predictions in the selection of candidate strains, the effectiveness of current influenza vaccines still fluctuates significantly, ranging from 20% to 60%. In addition, the effectiveness of vaccination in the elderly is lower, and appropriate adjuvants are needed to enhance its immunogenicity.

[0006] Therefore, a successful and effective influenza vaccine should include epitope information collected from multiple influenza variants so that the host immune system can produce broad protective immunity during the influenza season. The current influenza vaccine development strategy is to use nanoemulsion methods to deliver recombinant viral proteins / peptides to cover as many major epitopes as possible. Since the vaccine immunogenicity of proteins / peptides is low, adjuvants are generally co-encapsulated to solve this problem. However, a good emulsion formulation usually requires strong shear forces and the use of surfactants as stabilizers, which requires further purification to obtain an injectable formulation. This series of steps greatly increases the time and cost of vaccine synthesis. In addition, these methods also have the problems of low peptide loading and fast release, especially for water-soluble epitope peptides.

[0007] Therefore, designing a vaccine that can better cope with the high-frequency mutation of influenza viruses and train the body to produce specific immunity has important application value. However, how to effectively integrate the epitope peptide information of major virus subtypes is a key issue in vaccine design. Summary of the invention

[0008] Epitope peptide vaccines in the prior art have problems such as extremely difficult accurate identification and selection of effective epitopes, low immunogenicity of epitope peptides, easy degradation of epitope peptides by the body, and low stability. Based on this, the present invention provides a multi-epitope peptide composite vaccine based on the principle of self-assembly, and a preparation method and application thereof.

[0009] In the process of obtaining the scheme of the present application, the inventors of the present application found that some peptides extracted from SARS-CoV-2 structural proteins can self-assemble into nanocrystals with poly(I:C) (an exogenous nucleic acid adjuvant, Toll-like receptor 3 (TLR3) ligand), which is a spontaneous process driven by electrostatic and hydrophobic interactions. Machine learning analysis shows that these viral peptides have the same physicochemical characteristics as host antimicrobial peptides (AMPs), namely, cationic and amphiphilic. The poly(I:C) in the complex is not randomly distributed, but organized into liquid crystals with periodic microstructures, with an average spacing of about 3.4-3.6 nanometers, which enables them to bind to TLR3 multivalently, thereby promoting the recruitment of TLR3 and causing strong activation of downstream inflammatory pathways. Although less than 0.3% of the viral proteome is encapsulated in nanocrystals, the transcriptome of cells treated with nanocrystals is very consistent with the gene expression pattern of COVID-19.

[0010] Based on the above findings, the present application designs nanocrystals containing viral peptides into an influenza vaccine, which can then induce the body to produce broad-spectrum immune protection against major influenza variants.

[0011] In view of the difficulties in designing epitope peptide vaccines in the above prior art, this application designs a new vaccine formulation that can achieve the co-delivery effect of multiple epitope peptides. The scheme of this application can achieve the co-delivery of epitope peptides and nucleic acid adjuvants, and the scheme of this application can present multiple epitope peptides at one time, thereby improving the effectiveness and broad spectrum of the vaccine. At the same time, in specific applications, this application chooses to use influenza virus as a model to study the relationship between the structure of the assembly and the immune stimulation effect.

[0012] The purpose of the present invention can be achieved by the following technical solutions:

[0013] The present invention provides a multi-epitope peptide composite vaccine based on the self-assembly principle, comprising a viral antigenic epitope peptide and a nucleic acid adjuvant, wherein the viral antigenic epitope peptide and the nucleic acid adjuvant self-assemble to form nanocrystals with a periodic microstructure, wherein the periodic microstructure formed by a single epitope peptide and a nucleic acid adjuvant is called a single polypeptide nanocrystal, and the periodic microstructure formed by two or more composite epitope peptides and nucleic acid adjuvants is called a composite polypeptide nanocrystal.

[0014] The multi-epitope peptide composite vaccine based on the self-assembly principle of the present invention uses polypeptides as specific antigens to activate the body's specific immune response; nucleic acid adjuvants are used to activate the body's natural immune system and enhance the immunogenicity of epitope peptides.

[0015] The particle size of the multi-epitope peptide composite vaccine based on the self-assembly principle of the present invention is in the hundreds of nanometers, so it is easily internalized by cells, increasing the probability of the multi-epitope peptide composite vaccine based on the self-assembly principle contacting intracellular nucleic acid receptors, thereby better activating the body's natural immune system.

[0016] In one embodiment of the present invention, the multi-epitope peptide composite vaccine based on the self-assembly principle is an influenza vaccine capable of covering multiple influenza virus subtypes, and the antigenic epitope peptides of the virus are selected as epitope peptides in HA proteins of multiple subtypes of influenza viruses.

[0017] In one embodiment of the present invention, the multi-epitope peptide composite vaccine based on the self-assembly principle is an influenza vaccine capable of covering multiple influenza virus subtypes, including epitope peptides and nucleic acid adjuvants in one or more HA proteins of H1N1, H3N2, and IBV subtypes, wherein the epitope peptides and nucleic acid adjuvants self-assemble to form nanocrystals, wherein the nanoscale crystals formed by a single polypeptide and a nucleic acid adjuvant in the HA proteins of H1N1, H3N2, and IBV subtypes are single polypeptide nanocrystals, and the nanoscale crystals formed by two or more polypeptides and a nucleic acid adjuvant in the HA proteins of H1N1, H3N2, and IBV subtypes are composite polypeptide nanocrystals.

[0018] In one embodiment of the present invention, the epitope peptide of the HA protein of the H1N1 virus is selected from one or a combination of H1a, H1b, and H1c, the amino acid sequence of polypeptide H1a is shown in SEQ ID NO.1, the amino acid sequence of polypeptide H1b is shown in SEQ ID NO.2, and the amino acid sequence of polypeptide H1c is shown in SEQ ID NO.3.

[0019] In one embodiment of the present invention, the epitope polypeptide of the HA protein of the H3N2 virus is selected from one or a combination of H3a, H3b, and H3c, the amino acid sequence of polypeptide H3a is shown in SEQ ID NO.4, the amino acid sequence of polypeptide H3b is shown in SEQ ID NO.5, and the amino acid sequence of polypeptide H3c is shown in SEQ ID NO.6.

[0020] In one embodiment of the present invention, the epitope polypeptide of the HA protein of the IBV virus is selected from one or a combination of Ba, Bb or Bc, the amino acid sequence of polypeptide Ba is shown in SEQ ID NO.7, the amino acid sequence of polypeptide Bb is shown in SEQ ID NO.8, and the amino acid sequence of polypeptide Bc is shown in SEQ ID NO.9.

[0021] In one embodiment of the present invention, the nucleic acid adjuvant is dsRNA.

[0022] In a more preferred embodiment of the present invention, the nucleic acid adjuvant is poly(I:C). Poly(I:C) is polyinosinic acid-cytidylic acid, which is a synthetic dsRNA adjuvant commonly used in vaccine preparation. The embodiments of the present invention are also described by taking poly(I:C) as an example. Those skilled in the art can replace other nucleic acid adjuvants when implementing the scheme of the present invention, especially without departing from the core idea of ​​the present invention.

[0023] In one embodiment of the present invention, when the nucleic acid adjuvant is poly(I:C), the polypeptide and poly(I:C) are mixed at a charge ratio of 2.

[0024] In one embodiment of the present invention, when the antigenic epitope peptides of a virus are selected in two or a combination of two, the antigenic epitope peptides of different viruses are mixed in an equimolar ratio.

[0025] In one embodiment of the present invention, the multi-epitope peptide composite vaccine based on the self-assembly principle is an influenza vaccine capable of covering multiple influenza virus subtypes, and one of the following is selected:

[0026] (1) comprising an epitope polypeptide H1b of the HA protein of the H1N1 virus, an epitope polypeptide H3a of the HA protein of the H3N2 virus, and a nucleic acid adjuvant poly(I:C), wherein the polypeptide H1b, the polypeptide H3a, and the nucleic acid adjuvant poly(I:C) are self-assembled to form a composite polypeptide nanocrystal, wherein the polypeptide H1b and the polypeptide H3a are mixed in an equal molar ratio, and the total amount of the composite polypeptide and the poly(I:C) are mixed at a charge ratio of 2;

[0027] (2) comprising an epitope polypeptide H1b of the HA protein of the H1N1 virus, an epitope polypeptide H3c of the HA protein of the H3N2 virus, and a nucleic acid adjuvant poly(I:C), wherein the polypeptide H1b, the polypeptide H3c, and the nucleic acid adjuvant poly(I:C) self-assemble to form a composite polypeptide nanocrystal, wherein the polypeptide H1b and the polypeptide H3c are mixed in an equal molar ratio, and the total amount of the composite polypeptide and the poly(I:C) are mixed at a charge ratio of 2;

[0028] (3) comprising an epitope polypeptide H1c of the HA protein of the H1N1 virus, an epitope polypeptide Bc of the HA protein of the IBV virus, and a nucleic acid adjuvant poly(I:C), wherein the polypeptide H1c, the polypeptide Bc, and the nucleic acid adjuvant poly(I:C) self-assemble to form a composite polypeptide nanocrystal, wherein the polypeptide H1c and the polypeptide Bc are mixed in an equal molar ratio, and the total amount of the composite polypeptide and the poly(I:C) are mixed at a charge ratio of 2;

[0029] (4) It includes an epitope polypeptide H1a of the HA protein of the H1N1 virus, an epitope polypeptide Ba of the HA protein of the IBV virus, an epitope polypeptide Bc of the HA protein of the virus IBV and a nucleic acid adjuvant poly(I:C), wherein the polypeptide H1a, the polypeptide Ba, the polypeptide Bc and the nucleic acid adjuvant poly(I:C) self-assemble to form a composite polypeptide nanocrystal, wherein the polypeptide H1a, the polypeptide Ba and the polypeptide Bc are mixed in equal molar ratios, and the total amount of the composite polypeptide and the poly(I:C) are mixed at a charge ratio of 2.

[0030] The present invention further provides a method for preparing a multi-epitope peptide composite vaccine based on the self-assembly principle, comprising the following steps:

[0031] Dissolving the polypeptide lyophilized powder in a solvent to prepare a polypeptide stock solution;

[0032] dissolving the nucleic acid adjuvant in a buffer to prepare a nucleic acid adjuvant solution;

[0033] The polypeptide stock solution and the nucleic acid adjuvant solution are mixed and vortexed to obtain a multi-epitope peptide composite vaccine based on the principle of self-assembly, wherein the total amount of the polypeptide and the adjuvant are mixed at a charge ratio of 2 to form an Ampitope-poly (I: C) complex. The polypeptide stock solution and the nucleic acid adjuvant solution are mixed and vortexed to obtain a multi-epitope peptide composite vaccine based on the principle of self-assembly, wherein the total amount of the polypeptide and the nucleic acid adjuvant are mixed at a charge ratio of 2. When a single polypeptide is used, the single polypeptide and the nucleic acid adjuvant form a single polypeptide nanocrystal with a periodic microstructure, and when two or more composite polypeptides are used, the composite polypeptide and the nucleic acid adjuvant form a composite polypeptide nanocrystal with a periodic microstructure; when a single polypeptide or a composite polypeptide and the nucleic acid adjuvant cannot form a nanocrystal with a periodic microstructure, this type of complex is called a nanocomplex.

[0034] In one embodiment of the present invention, the solvent is selected from dimethyl sulfoxide (DMSO).

[0035] In one embodiment of the present invention, the buffer is a 10 mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer at pH 7.4, which contains 140 mM NaCl.

[0036] The present invention further provides an application of a multi-epitope peptide composite vaccine based on the self-assembly principle, and an application of the multi-epitope peptide composite vaccine based on the self-assembly principle in the preparation of medicines.

[0037] The present invention explores a new vaccine formulation that can achieve co-delivery of multiple epitope peptides. The core of this method is the self-assembly of viral epitope peptides and nucleic acid adjuvants (poly(I:C)), which act as natural adjuvants to enhance immune responses. This application focuses on a special class of epitope peptides, namely polypeptides that have the same physicochemical characteristics as host antimicrobial peptides (AMPs). These polypeptides are positively charged and amphiphilic, and can be assembled into nanocrystals with periodic microstructures through electrostatic attraction with nucleic acid adjuvants. Since nanocrystals with periodic microstructures can significantly induce the recruitment and activation of nucleic acid receptors of host cells, multiple epitope peptides can be integrated into the same nanocrystal, so this solution simultaneously improves the immunogenicity and broad spectrum of polypeptide vaccines.

[0038] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0039] 1. The present application proposes a new epitope peptide screening method. The screened epitope peptides have physical and chemical properties similar to those of antimicrobial peptides. Therefore, they have the ability to integrate nucleic acid adjuvants such as poly(I:C) into pro-inflammatory (promoting the release of inflammatory factors) nanocrystals. On the one hand, the co-delivery of antigens and poly(I:C) as a nucleic acid adjuvant is achieved, and on the other hand, the immune activation function of the nucleic acid adjuvant poly(I:C) can be enhanced.

[0040] 2. This application innovatively uses AMP-like epitope peptides to control the distance of poly(I:C) in the crystal, making it similar to the size of TLR3 nucleic acid receptors, which can induce large-scale aggregation and orderly arrangement of TLR3, thereby enhancing the cell response to poly(I:C) adjuvants. This application found that different AMP-like epitope peptides can assemble poly(I:C) into nanocrystals with different spacing, which can fine-tune the level of immune activation to meet specific clinical needs.

[0041] 3. This application innovatively utilizes the principle of "crystal structure compatibility" of AMP-like epitope peptides, so that multiple AMP-like epitope peptides can be assembled into the same crystal. This co-assembly principle allows those T cell epitope peptides that have weak binding effects with poly(I:C) to be integrated into the crystal, thereby broadening the broad spectrum of epitope peptide delivery. For example, in the composite polypeptide nanocrystals made of H1a, Ba and Bc composite polypeptides and poly(I:C), only Bc can organize poly(I:C) into a columnar lattice with an average poly(I:C) spacing of 3.67 nanometers, while the other two epitope peptides cannot induce poly(I:C) to form a periodic microstructure. Therefore, the crystal structure of the ternary AMP-like epitope peptide system is very similar to the crystal structure made using Bc (the average poly(I:C) spacing is 3.76 nanometers), and the area of ​​the ordered region is larger. In contrast, in the study of nanocrystals or nanocomplexes made of H1b, H3b and Bc with poly(I:C), although these three peptides can organize poly(I:C) into columnar crystals, the crystal structures are different. The average spacing of poly(I:C) in H1b-poly(I:C) nanocrystals is 4.4 nanometers. The average spacing of poly(I:C) in H3b-poly(I:C) nanocrystals is 3.9 nanometers. The spacing of Bc-poly(I:C) nanocrystals is 3.67 nanometers. At this time, the multiple epitope peptides involved in the formation of composite polypeptide nanocrystals have different crystal structures formed with poly(I:C), and cannot synergistically induce the generation of periodic microstructures. These results indicate that when integrating poly(I:C), the crystal structure compatibility between the participating AMP-like epitope peptides determines the formation of composite polypeptide nanocrystals.

[0042] The present invention provides a promising method for designing universal vaccines (e.g., universal influenza vaccines). The method co-encapsulates the antigenic epitope peptide of the viral HA protein and an exogenous nucleic acid adjuvant (poly(I:C)) into nanocrystals to form a new type of vaccine. The vaccine can improve the stability of viral antigens and the efficiency of antigen delivery, improve the reduced immune effect caused by antigen drift in traditional processes, and can simultaneously activate strong natural immune responses and acquired immune responses (cellular immunity), and has better broad spectrum. The vaccine preparation process is simple and has strong application feasibility.

[0043] Although the present invention is described in detail using the preparation of influenza vaccines as an example, the scheme of the present application has broader application potential, including vaccines for other infectious diseases and cancers, providing breadth and depth for vaccine-induced protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 To identify AMP-like epitopes (Ampitopes) in the hemagglutinin (HA) proteins of three representative influenza viruses based on machine learning.

[0045] Figure 2 The results of identification of AMP epitopes of hemagglutinin (HA) proteins of three subtypes of influenza viruses based on machine learning.

[0046] Figure 3 The results of correlation analysis between the main physicochemical characteristics of Ampitopes and the size of nanocrystals or nanocomplexes.

[0047] Figure 4 The results of structural characterization of Ampitope(s)-poly(I:C) nanocrystals or nanocomposites using small-angle X-ray scattering (SAXS).

[0048] Figure 5 Ampitope(s)-poly(I:C) nanocomplexes and nanocrystals induce immune activation in macrophages.

[0049] Figure 6 Ampitope(s)-poly(I:C) nanocrystals activate inflammatory responses in C57BL / 6J mice.

[0050] Figure 7 This is the result of Ampitope-poly(I:C) nanocrystals activating cellular immune response.

[0051] Figure 8 This is a schematic diagram of the preparation principle of the multi-epitope peptide composite vaccine based on the self-assembly principle of the present invention. DETAILED DESCRIPTION

[0052] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0053] Example 1

[0054] The epitope peptides of the HA protein of representative influenza virus strains are determined by the following specific method:

[0055] 1. Selection of HA proteins of representative influenza virus strains

[0056] In this example, HA proteins of three representative influenza virus strains were selected, and HA proteins of one strain from each subtype were selected as the target of subsequent research, A / California / 07 / 2009 (H1N1) that caused the 2009 H1N1 influenza pandemic, A / Hong Kong / 1-11-MA21-1 / 1968 (H3N2) that caused the 1968 influenza pandemic, and B / Hong Kong / 22 / 2001, an influenza B virus strain representing seasonal influenza.

[0057] In this example, A / California / 07 / 2009 (H1N1, GCA_001343785.1), A / Hong Kong / 1-11-MA21-1 / 1968 (H3N2, ACF41779.1) and B / Hong Kong / 22 / 2001 (IBV, ABL77090.1) were selected. For simplicity, the HA proteins of the above strains will be referred to as influenza-H1, influenza-H3 and influenza-B, respectively. The HA proteins of representative influenza virus strains were selected in this example, which can cover a variety of influenza virus subtypes.

[0058] 2. Screen epitope peptides and identify AMP-like epitope peptides (Ampitopes)

[0059] (1) The amino acid sequences of three HA proteins (i.e., influenza-H1, influenza-H3, and influenza-B) were obtained from the Uniprot database, where the amino acid sequence length of the HA protein of influenza-H1 was 565, the amino acid sequence length of H3N2 was 566, and the amino acid sequence length of IBV was 378, for a total of 1509 amino acids.

[0060] (2) Use existing mature epitope prediction platforms (e.g., https: / / services.healthtech.dtu.dk / ) to screen peptide sequences. Use NetMHCcons to predict sequences that bind to MHC-I, and use NetMHCII to predict sequences that bind to MHC-II. Screen HA epitope peptides that can bind to major histocompatibility complex class I (MHC-I) or MHC-II from the HA proteins of three representative virus strains.

[0061] MHC-I and MHC-II are two membrane proteins on the surface of antigen presenting cells (APCs), responsible for T cell immunity and B cell immunity, respectively. Epitopes that bind strongly to HLA-C1 (MHC-I) will be presented to T cells and induce good cellular immunity, while epitopes that bind strongly to HLA-DR (MHC-II) will be presented to B cells and induce strong humoral responses. Since T cell-mediated cellular immunity, especially CD8+T cell responses, play a key role in controlling and eliminating viral infections, epitope peptides that bind strongly to MHC-I are more concerned.

[0062] Figure 1 These are the results of the identification of AMP-like epitope peptides (Ampitopes) of the hemagglutinin (HA) proteins of three influenza virus subtypes based on machine learning. Figure 1 AC shows the predicted HA epitopes binding to HLA-C1 molecules (strong binding and weak binding). Figure 1 The horizontal axes of A, B, and C are the positions of amino acids. Since the positions of the selected peptides do not exceed 500, the horizontal axes select part of the length; Figure 1 The vertical coordinates of A, B, and C are the 12 most common HLA-C1 alleles. Figure 1 DF shows the predicted HA epitopes that bind (strongly bind) to HLA-DR molecules. Figure 1 The horizontal axes of D, E, and F are the positions of amino acids. Since the positions of the selected peptides do not exceed 500, the horizontal axes select part of the length; Figure 1 The vertical axes of D, E, and F represent the six most common HLA-DR alleles. Figure 1 In the figure, blocks of different colors represent the positions of the predicted polypeptides that can bind to HLA molecules in the prediction results of different alleles.

[0063] The results showed that both T-cell and B-cell epitopes were abundant and evenly distributed throughout the HA protein ( Figure 1 AF).

[0064] (3) Excluding repeated sequences, a pre-trained SVM-based AMP classifier was used to identify AMP-like peptides in HA protein. Each query sequence has a σ score. The first amino acid in each sequence is colored according to the score to reflect whether the query sequence is an AMP. The positive and negative σ scores represent the probability of whether the sequence is an AMP (P(+1)), respectively. "P" stands for probability, i.e. Probability, and the brackets represent "conditions". P(+1) represents the probability that the peptide is an AMP, and P(-1) represents the probability that the peptide is not an AMP, P(-1) = 1–P(+1). P is a logistic regression of the SVM output score (σ), which is fitted by additional cross-validation of the training data. The specific method is platt scaling. This method is a known method, which can be referred to in the literature: Mapping membrane activity in undiscovered peptide sequence space using machine learning, the link is https: / / www.pnas.org / doi / full / 10.1073 / pnas.1609893113#sec-2. σ score > 0 corresponds to P(+1) > 0.5. The greater the positive value of σ score, the higher the possibility of being an AMP. Sequences with σ < 0 (non-AMP) are marked in dark blue. See the specific results for details. Figure 1 GI. Detailed information about the SVM model, including training data sets, physicochemical descriptors and training methods, is prior art, see the literature: MW Lee, EY Lee, ALFerguson, GCL Wong, Machine learning antimicrobial peptide sequences: Some surprising variations on the theme of amphiphilic assembly, Curr Opin Colloid Interface Sci, 2018, 38, 204-213.

[0065] HA epitope peptides with physicochemical characteristics similar to those of natural antimicrobial peptides (AMPs) were screened from HA proteins of three representative virus strains. They are able to assemble poly(I:C) into nanocrystals or nanocomplexes, thereby strongly activating TLR3-mediated inflammatory responses.

[0066] The results were consistent with expectations, with AMP-like peptide sequences clustering into hotspots and varying distributions in the selected HA proteins. In influenza-H1, two major AMP hotspots appeared in the receptor binding site (RBS) of HA1 and the helical region of HA2 ( Figure 1A). In contrast, in influenza-H3 and influenza-B, the two strong AMP hotspots previously observed in influenza-H1 have disappeared. However, a new hotspot has appeared at the C-terminus of the HA1 protein in both influenza virus strains ( Figure 1 B). In influenza-B, two new hot spots appeared at the N-terminus of the HA1 subunit. The hot spot of HA-2 did not completely disappear, but the brightness was significantly reduced ( Figure 1 C).

[0067] A large-scale screening of 4935 HA proteins collected from various subtypes showed that the distribution of AMP hotspots and AMP scores were relatively conserved among strains classified as the same subtype ( Figure 2 ).

[0068] (4) Based on the σ score and epitope prediction, three epitope peptides were selected from the HA protein of each influenza virus strain, namely, Ampitopes. Figure 1 In AC, the polypeptide indicator arrows described by JL are located above the HLA-C1 prediction map to determine the position of the selected sequence. "μ" is the average σ score of all homologous sequences in this influenza subtype. The calculation method of "μ" is: find the homologous sequences of the sequence in the 4935 collected HA proteins, screen these sequences using the AMP screening model and score "σ", and then take the average of these scores, which is "μ". It is used to evaluate the conservation of the AMP characteristics of this site. The "hit rate" of each sequence is calculated by dividing the number of HLA-C1 / HLA-DR alleles bound by the sequence by the number of all alleles included in the prediction. The last two columns summarize the results.

[0069] Three HA epitope peptides that can bind to MHC-I or MHC-II and have similar physical and chemical characteristics to AMP were selected from each of the three representative virus strains, called Ampitopes ( Figure 1 At the same time, large-scale data analysis showed that the AMP properties of the selected Ampitopes were relatively conserved in each subtype, indicating that the mutations may not affect the condensation activity of poly(I:C).

[0070] 3. Peptide synthesis

[0071] The amino acid sequences of all polypeptides (H1a, H1b, H1c, H3a, H3b, H3c, Ba, Bb and Bc) obtained according to the above steps are shown in Table 1.

[0072] Table 1. Names of polypeptides involved in this example and their amino acid sequences

[0073]

[0074] Among them, the amino acid sequence of polypeptide H1a is shown in SEQ ID NO.1, the amino acid sequence of polypeptide H1b is shown in SEQ ID NO.2, the amino acid sequence of polypeptide H1c is shown in SEQ ID NO.3, the amino acid sequence of polypeptide H3a is shown in SEQ ID NO.4, the amino acid sequence of polypeptide H3b is shown in SEQ ID NO.5, the amino acid sequence of polypeptide H3c is shown in SEQ ID NO.6, the amino acid sequence of polypeptide Ba is shown in SEQ ID NO.7, the amino acid sequence of polypeptide Bb is shown in SEQ ID NO.8, and the amino acid sequence of polypeptide Bc is shown in SEQ ID NO.9, wherein H1a, H1b, and H1c are three epitope polypeptides of the HA protein of H1N1 virus, H3a, H3b, and H3c are three epitope polypeptides of the HA protein of H3N2 virus, and Ba, Bb, and Bc are three epitope polypeptides of the HA protein of IBV virus.

[0075] All peptides (H1a, H1b, H1c, H3a, H3b, H3c, Ba, Bb, and Bc) were custom synthesized by Jier Biochemical (Shanghai) Co., Ltd. (purity >95% confirmed by HPLC), and the net charge of the specific peptides was calculated using Prot pi (https: / / www.protpi.ch / ). The linear charge density was calculated using the following formula:

[0076] Linear charge density = net charge of a given sequence / total amount of amino acids. The hydrophobicity of each sequence was calculated using the Eisenberg scale.

[0077] Example 2

[0078] The preparation of Ampitope(s)-poly(I:C) nanocrystals or nanocomposites is as follows:

[0079] The purpose of this example is to explore whether the selected Ampitopes can condense poly(I:C) into nanocrystals with spacing within the optimal spacing range, and whether Ampitopes in different influenza virus strains can synergistically assemble poly(I:C) into nanocrystals with periodic microstructures.

[0080] Studies have shown that AMP-like polypeptides can organize nucleic acids into nanocrystals, allowing nucleic acids to bind to nucleic acid receptors such as TLR3, TLR9, etc. in a multivalent form. The spacing between nucleic acids (the optimal spacing is 3-4 nanometers) determines the amplitude of TLR-mediated immune activation. The deviation of the nucleic acid spacing from the optimal spacing will result in a decrease in the degree of immune activation. In order to explore whether the selected Ampitopes can condense poly(I:C) into nanocrystals with spacing within the optimal spacing range, and whether Ampitopes in different influenza virus strains can synergistically assemble poly(I:C) into nanocrystals with periodic microstructures, this example solves this series of problems through dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS) experiments.

[0081] 1. Prepare Ampitope(s)-poly(I:C) nanocrystals or nanocomposites by:

[0082] The peptide stock solutions were prepared by dissolving the lyophilized powders of peptides (H1a, H1b, H1c, H3a, H3b, H3c, Ba, Bb and Bc) in dimethyl sulfoxide (DMSO) at a concentration of 10 mg / mL for each peptide. ploy(I:C) (high molecular weight, commercialized double-stranded RNA, from InvivoGen, catalog number tltl-pic) was dissolved in 10 mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer (containing 140 mM NaCl) at pH 7.4 at a concentration of 5 mg / mL. Ampitope(s) and poly(I:C) are mixed at a charge ratio of 2 (the specific mixing method is: first prepare them into polypeptide or composite polypeptide solutions respectively, then calculate the average charge of the polypeptide in the solution, and then mix them at a charge ratio of 2 between the polypeptide (or composite polypeptide) and poly(I:C); in the mixed solution, the polypeptide (or composite polypeptide) can assemble poly(I:C) into nanocrystals with a periodic microstructure), wherein Ampitope(s) refers to a single polypeptide or two composite polypeptides (at a molar mass ratio of 1:1) or three composite polypeptides (at an equal molar ratio) to form an Ampitope(s)-poly(I:C) complex. The complexes of single Ampitope (H1a, H1b, H1c, H3a, H3b, H3c, Ba, Bb or Bc) and poly(I:C), the complexes of double Ampitopes (H1b+H3a, H1b+H3c, H1c+Bc) and poly(I:C), and the complexes of three Ampitopes (H1a+Ba+Bc) and poly(I:C) were prepared respectively. The mixture of double Ampitopes was prepared by mixing two Ampitopes at a molar ratio of 1:1, and the mixture of three Ampitopes was prepared by mixing three Ampitopes at a molar ratio of 1:1:1.

[0083] 2. Dynamic Light Scattering (DLS) Measurement

[0084] The above nanocomplexes (see Table 2) were added to ultrapure water at a ratio of 1:100 (v / v), and then the particle size and polydispersity index (PDI) of the complexes were detected using DLS, and then the diameter or PDI was plotted against three relevant physicochemical properties (net charge, linear charge density, and hydrophobicity). The correlation index (CI) was calculated using the Pearson correlation function.

[0085] Figure 3 Correlation analysis between the main physicochemical characteristics of Ampitopes and the size of nanocomposites. Figure 3AC takes values ​​between 0 and 1 to indicate the strength of the correlation, with positive values ​​indicating positive correlation and negative values ​​indicating negative correlation, and a trend line is added to show the correlation more intuitively. Figure 3 In , A refers to the relationship between the net charge number of the polypeptide and the particle size and polydispersity index of the complex; Figure 3 In , B is the relationship between the charge density of the polypeptide (net charge / number of polypeptide amino acids) and the particle size and polydispersity index of the complex; Figure 3 In the equation, C is the relationship between the hydrophobicity of the peptide and the particle size and polydispersity index of the complex.

[0086] Similar to AMPs, all individual Ampitopes were able to condense poly(I:C) into nanocomplexes with diameters ranging from 199 to 3485 nm. Five of the nanocomplexes (H1a-poly(I:C), H1b-poly(I:C), H1c-poly(I:C), Bb-poly(I:C), Bc-poly(I:C)) had diameters less than 400 nm, which is an ideal size range for cellular uptake and entry of TLR3 into endosomes (see Table 2). Among the selected Ampitopes, H1a, H1b, Bb, and Bc were the ones with the strongest ability to produce nanocomplexes with good size and size distribution. Pearson correlation analysis showed that Ampitopes with lower hydrophobicity and higher net charge and linear charge density tended to condense poly((I:C) into complexes with smaller diameters and narrower size distributions. The results indicate that the cationic charge and hydrophilicity of Ampitopes have a great influence on the formation of stable electrostatic complexes (see Figure 3 AC).

[0087] Table 2 Hydrodynamic dimensions of poly((I:C) nanocomplexes composed of mono-, di- and tri-epitope peptides

[0088]

[0089]

[0090] In Table 2, the horizontal grids with filled colors are the types of polypeptides used in the nanocomplex, such as the polypeptides used in the three Ampitopes are H1a, Ba, and BC. In Table 2, * refers to influenza (Flu).

[0091] 3. Transmission electron microscopy (TEM) observation

[0092] The nanocrystal sample was dispersed in ultrapure water to make a suspension, and then the suspension was dropped on a carbon-coated copper grid (300 mesh), and ammonium molybdate negative staining solution was added and incubated for 10 seconds. The remaining liquid was absorbed with filter paper and dried at room temperature for 1 hour. TEM observation was performed using a 120kv Talos L120C G2 microscope. The image was captured by a Ceta CMOS camera, and the TEM image was analyzed using ImageJ software.

[0093] 4. Small-angle X-ray scattering (SAXS) analysis

[0094] In this section, SAXS was used to analyze the crystal structure of Ampitope(s)-poly(I:C) nanocomplexes.

[0095] The Ampitope(s)-poly(I:C) nanocomplexes in Table 2 were equilibrated at room temperature for about 1 week and transferred to 1.5 mm quartz capillaries (Quarzkapillaren, Mark-tubes). SAXS measurements were performed at the Shanghai Synchrotron Radiation Source (SSRF, BL19U2 beamline) using monochromatic X-rays with a threshold of 6 keV. The scattered radiation was measured using a Pilatus 1M, Dectris detector (detector plane 0.33 mm (horizontal) × 0.05 mm (vertical)). To ensure data consistency, independent and identical samples were prepared and measured at multiple time points. The two-dimensional (2D) powder diffraction pattern was integrated by BioXTAS RAW. The SAXS data were analyzed by plotting the integrated scattering intensity I(q) versus momentum transfer q using BioXTAS RAW. The peak positions were measured by Lorentzian fitting of the diffraction peaks. The structure of the complex was determined based on the ratio between the q positions of all recorded peaks and compared with the allowed reflectivity of the known liquid crystal phase (the Lorentz fitting was performed in MATLAB, and the specific method can be found in this webpage:

[0096] https: / / www.mathworks.com / matlabcentral / fileexchange / 33775-lorentzfit-xy-varar gin). The position of the first diffraction peak is used to calculate the average distance between poly(I:C), and the calculation formula is d = 2π / q.

[0097] The results are as follows Figure 4 As shown in Figure 2, in a single Ampitope system, a single diffraction peak was observed in the complexes prepared with epitope peptides of H1b, Bb, or Bc, which could be divided into a two-dimensional (2D) columnar lattice. The position of the first diffraction peak was between 0.142 and The corresponding spacing of poly(I:C) is 3.5 to 4.4 nanometers ( Figure 4 A). No diffraction peak was observed for H1a, and its sequence may be too short to organize the orderly arrangement of poly(I:C). This result is also consistent with the results of previous DLS measurements to a certain extent.

[0098] In a dual-Ampitopes system, it was investigated whether Ampitopes with different poly(I:C) aggregation activities could synergistically promote the growth of immunostimulatory nanocrystals. Based on DLS and SAXS measurements, H3a alone could not effectively condense poly(I:C) into nanocrystals, but when H1b (a good poly(I:C) aggregation Ampitope) was introduced, the two Ampitopes began to co-assemble with poly(I:C) into nanocrystals with tighter spacing (spacing in the crystals was 3.8 nm, while the spacing in the H1b-poly(I:C) complex was 4 nm), close to the optimal spacing for TLR3 activation (activity was maximized when the spacing of poly(I:C) was 3.4 nm). The diffraction peaks were also sharper than those of H1b-poly(I:C) nanocrystals, indicating that there was a synergistic effect between the Ampitopes that promoted the formation of more ordered structures, such as Figure 4 B.

[0099] Similar trends were also observed in the three-ampitope system containing H1a, Ba, and Bc. In the three-ampitope system, H1a and Ba were unable to condense poly(I:C) into nanocrystals when acting alone. However, when Bc (a good poly(I:C) condensation ampitope) was introduced into the mixture of H1a and Ba and then incubated with poly(I:C), the resulting nanocrystals also showed a more ordered structure than the nanocrystals formed by Bc alone with poly(I:C) ( Figure 4 C, E). Transmission electron microscopy (TEM) images show that the size of the Bc-poly(I:C) complex is approximately 200 nanometers ( Figure 4 D). Given that not all key epitopes possess the AMP-like physiochemical features necessary for effective binding to poly(I:C), synergy between these ampitopes is important for the development of universal influenza vaccines that can deliver multiple epitopes.

[0100] Example 3

[0101] To investigate the activation of antigen presenting cells (APCs) induced by Ampitope nanocrystals or nanocomplexes.

[0102] 1. In vitro immune stimulation experiment

[0103] Treatment of RAW-Lucia with monomeric poly(I:C), Ampitope(s)-poly(I:C) nanocrystals, or Ampitope(s)-poly(I:C) nanocomplexes TM ISG cells.

[0104] RAW-Lucia involved in this embodiment TM ISG cells are a mouse macrophage cell line carrying a luciferase reporter gene and are a known commercial cell.

[0105] The culture medium involved in this embodiment is a complete culture medium, namely Dulbecco's modified Eagle's medium (DMEM) containing 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin-streptomycin.

[0106] One day before stimulation, cells were seeded into 96-well plates (3×10^4 cells per well) and cultured with complete medium. Subsequently, cells were stimulated for 24 hours with complexes containing ploy(I:C) (2.5μg / mL) and single peptides or complex peptides (two or three peptides mixed in an equimolar ratio). The PBS-treated group was used as a negative control to exclude the background of non-complex stimulation.

[0107] To evaluate the stimulated immune activity, luciferase expression in the cell supernatant was measured 24 h later using a luciferase assay reagent. The fold activation (FOA) was calculated by normalizing the luciferase signal in sample-treated cells with that in PBS-treated cells. All experiments were repeated three times. Data are presented as mean ± standard error. Statistical analysis was performed using a t-test (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0108] Compared with nanocrystals, poly(I:C) in nanocomposites does not have a spatially periodic distribution.

[0109] The results showed that Ampitope itself hardly activated macrophages, indicating that the immunogenicity of the epitope peptide was quite low (activation fold was about 1.4). In contrast, poly(I:C) treatment activated macrophages 126 times more than PBS treatment ( Figure 5 A). Figure 5 In the table, pIC indicates poly(I:C) alone.

[0110] In addition, four ampitopes that can condense poly(I:C) into ampitopes with good TLR3 pathway activation, namely H1a, H1b, Bb, and Bc, were also studied in detail.

[0111] Except for H1a, the remaining Ampitopes (H1b, Bb and Bc) can condense poly(I:C) into nanocrystals. The results showed that single Ampitope nanocrystals composed of H1b, Bb or Bc and poly(I:C) (when different polypeptide Ampitope(s) and poly(I:C) are complexed, they are also mixed with a charge ratio of 2) induced 238 times, 263 times and 222 times of activation, respectively, while H1a and poly(I:C) could not form nanocrystals with periodic microstructures. However, the particle size of the complex of H1a and poly(I:C) is also at the nanometer level, so it is called H1a-poly(I:C) nanocomplex. The activation induced by the H1a-poly(I:C) nanocomplex is slightly lower, at 186 times, which is 22.8% lower than the average activation level of the three nanocrystal complexes, indicating that nanocrystals with periodic microstructures enable the multivalent presentation of poly(I:C) to have a positive effect on TLR3-mediated immune activation ( Figure 5 A). In addition, the composite Ampitopes nanocrystals formulated with H1b+H3a induced 234-fold activation, while the composite Ampitopes nanocrystals formulated with H1a+Ba+Bc induced 221-fold activation ( Figure 5 B, C). Both formulations activated macrophages much more strongly than the poly(I:C) control, but at the same level as Ampitope nanocrystals alone.

[0112] 2. Animal experiments

[0113] Female C57BL / 6J mice aged 8 to 10 weeks were intramuscularly injected with ploy(I:C) (100 μg / mouse) and / or composite peptides (H1b+H3a, H1b+H3c, H1c+Bc, H1a+Ba+Bc, 344 μg / mouse, wherein the peptides were mixed at a molar ratio of 1:1 or 1:1:1), with a final injection volume of 200 μL. One hour after the injection of ploy(I:C) and / or composite peptides, 200 μL of blood samples were collected by puncture of the mandibular venous plexus and collected in anticoagulation tubes. Plasma was prepared by centrifugation at 6000×g and 4°C for 10 minutes. The IL-6 and IFN-α levels in plasma were quantified by ELISA, and plasma collected from untreated mice was used to assess the baseline levels of IL-6 and IFN-α. This experiment used n=5 (5 mice per experimental group). Elispot method was used for cellular immunity detection, and the cellular immunity level was detected by counting the spots. Statistical analysis was performed using t test (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). All experimental animals were approved by the Animal Care and Use Committee (IACUC) of Westlake University.

[0114] This section examines the release of two key cytokines, IL-6 and IFN-α. IL-6 is an early inflammatory marker that is released by macrophages and myocytes in skeletal muscle fibers, serving as a recruitment and activation signal for other immune cells. In contrast, IFN-α is primarily secreted by plasmacytoid dendritic cells, which activates interferon-stimulated genes (ISGs) in a variety of cell types, inducing the expression of downstream antiviral effector molecules.

[0115] Mice were injected intramuscularly with double Ampitopes nanocrystals (composite peptide nanocrystals formed by H1b+H3a, H1b+H3c or H1c+Bc and poly(I:C)) and triple Ampitopes nanocrystals (composite peptide nanocrystals formed by H1a+Ba+Bc and poly(I:C)). PBS, composite Ampitopes and poly(I:C) were injected as control groups. Since IL-6 and IFN-α have a shorter cycle in the inflammatory response, plasma samples were collected 1 hour after injection and the levels of these two cytokines were analyzed ( Figure 6 A).

[0116] Consistent with the in vitro results, injection of Ampitopes had minimal effect on elicited cytokine release. In contrast, injection of poly(I:C) induced a 33-fold increase in IL-6 release and a 70-fold increase in IFN-α release, suggesting that poly(I:C) has a strong adjuvant effect. However, the extent of immune activation in poly(I:C)-treated mice varied widely, ranging from 12 to 2589 pg / mL for IL-6 and 29 to 5426 pg / mL for IFN-α. This phenomenon may be due to the different cellular uptake and tissue retention of free poly(I:C).

[0117] When poly(I:C) was condensed into nanocrystals by Ampitopes, the heterogeneity of the data was significantly reduced. More importantly, when poly(I:C) was condensed into nanocrystals by Ampitopes, the release of IL-6 was increased by 100 times, which was better than the effect after poly(I:C) treatment ( Figure 6 B, C, Figure 6 B is a combination of H1b+H3a). For IFN-α, it was found that the release of IFN-α increased by 78 times ( Figure 6 E), which was comparable to poly(I:C) treatment alone. However, dual Ampitopes treatment resulted in only a 15-fold increase ( Figure 6 D, Figure 6 B, which is a combination of H1b+H3a), was lower than the 70-fold increase in the poly(I:C)-treated group, indicating an inhibitory effect.

[0118] The combined in vivo data suggest that delivering ampitopes in nanocrystal form helps improve the immunogenicity of the ampitopes. The inflammatory responses elicited by the nanocrystals were more consistent, more predictable, and in some cases stronger than adjuvant alone.

[0119] At the same time, the cellular immunity induced by nanocrystals was detected. The ELISpot method was used to detect the secretion of cytokines interleukin 2 (IL-2) and interferon gamma (IFN-γ), which is a commonly used cellular immunity detection method. Among them, IFN-γ can induce CD4+T cells to differentiate into Th1 (T helper type 1 cells), and Th1 mainly mediates cellular immune response; and IL-2 secreted by Th1 can change the differentiation balance of Th1 and Tfh (follicular helper T cells), making it more favorable to Th1, and is conducive to the expansion of CD8 CTL cell populations, which is conducive to the targeted elimination of intracellular viruses.

[0120] The results are as follows Figure 7 As shown, compared with the injection of poly(I:C) alone, the injection of the complex polypeptide alone or the injection of PBS alone, the nanocrystal group was able to significantly activate the secretion of IL-2 and IFN-γ. It can be inferred that the nanocrystals can induce specific cellular immune responses and enhance the body's targeted clearance of intracellular infectious viruses.

[0121] The above describes the scheme of the present invention through specific embodiments. Figure 8 This is a schematic diagram of the preparation principle of the multi-epitope peptide composite vaccine based on the self-assembly principle of the present invention.

[0122] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A multi-epitope peptide composite vaccine based on the principle of self-assembly, characterized in that: It includes viral antigenic epitope peptides and nucleic acid adjuvants, which self-assemble to form nanocomplexes without periodic microstructures or nanocrystals with periodic microstructures. The nanocrystals include two types, one is single polypeptide nanocrystals formed by a single epitope peptide and a nucleic acid adjuvant, and the second is composite polypeptide nanocrystals formed by two or more epitope peptides and nucleic acid adjuvants. The multi-epitope peptide composite vaccine based on the self-assembly principle uses polypeptides as specific antigens to activate the body's specific immune response. The nucleic acid adjuvant is used to activate the body's natural immune system and enhance the immunogenicity of the epitope peptides.

2. A multi-epitope peptide composite vaccine based on the self-assembly principle according to claim 1, characterized in that: The arrangement of the nucleic acid adjuvant in the nanocrystal with a periodic microstructure formed by self-assembly of the antigenic epitope peptide of the virus and the nucleic acid adjuvant is periodic, and the average spacing of the nucleic acid adjuvant is 3.4-3.6 nanometers.

3. A multi-epitope peptide composite vaccine based on the self-assembly principle according to claim 1, characterized in that: The particle size of the multi-epitope peptide composite vaccine based on the self-assembly principle is on the scale of hundreds of nanometers.

4. A multi-epitope peptide composite vaccine based on the self-assembly principle according to claim 1, characterized in that: The multi-epitope peptide composite vaccine based on the self-assembly principle is an influenza vaccine capable of covering multiple influenza virus subtypes, and the antigenic epitope peptides of the virus are selected as HA epitope polypeptides of influenza viruses of multiple subtypes.

5. A multi-epitope peptide composite vaccine based on the self-assembly principle according to claim 1, characterized in that: The multi-epitope peptide composite vaccine based on the self-assembly principle is an influenza vaccine that can cover multiple influenza virus subtypes, including one or two or more epitope peptides and nucleic acid adjuvants in H1N1, H3N2, and IBV subtype viruses, and the epitope peptides and nucleic acid adjuvants self-assemble to form nanocomplexes or nanocrystals, wherein the nanocomplex refers to an aperiodic microstructure formed by the epitope peptides of the HA protein in the H1N1, H3N2, and IBV subtypes and the nucleic acid adjuvants, and the nanocrystal refers to a periodic microstructure formed by the epitope peptides of the HA protein in the H1N1, H3N2, and IBV subtypes and the nucleic acid adjuvants.

6. A multi-epitope peptide composite vaccine based on the self-assembly principle according to claim 5, characterized in that: The epitope peptide of the HA protein in the H1N1 subtype is selected from one or a combination of H1a, H1b, and H1c, the amino acid sequence of the polypeptide H1a is shown in SEQ ID NO.1, the amino acid sequence of the polypeptide H1b is shown in SEQ ID NO.2, and the amino acid sequence of the polypeptide H1c is shown in SEQ ID NO.3; The epitope peptide of the HA protein in the H3N2 subtype of the protein is selected from one or a combination of H3a, H3b, and H3c, the amino acid sequence of the polypeptide H3a is shown in SEQ ID NO.4, the amino acid sequence of the polypeptide H3b is shown in SEQ ID NO.5, and the amino acid sequence of the polypeptide H3c is shown in SEQ ID NO.6; The epitope peptide of the HA protein in the IBV subtype of the protein is selected from one or a combination of Ba, Bb or Bc, the amino acid sequence of polypeptide Ba is shown in SEQ ID NO.7, the amino acid sequence of polypeptide Bb is shown in SEQ ID NO.8, and the amino acid sequence of polypeptide Bc is shown in SEQ ID NO.

9.

7. The multi-epitope peptide composite vaccine based on the self-assembly principle according to claim 5, characterized in that: The nucleic acid adjuvant is dsRNA.

8. The multi-epitope peptide composite vaccine based on the self-assembly principle according to claim 7, characterized in that: The nucleic acid adjuvant is poly(I:C), and the polypeptide and poly(I:C) are mixed at a charge ratio of 2.

9. The multi-epitope peptide composite vaccine based on the self-assembly principle according to claim 5, characterized in that: When the antigenic epitope peptides of viruses are selected in two or a combination of two, the antigenic epitope peptides of different viruses are mixed in an equimolar ratio.

10. The multi-epitope peptide composite vaccine based on the self-assembly principle according to claim 5, characterized in that: The multi-epitope peptide composite vaccine based on the self-assembly principle is an influenza vaccine that can cover multiple influenza virus subtypes, and one of the following is selected: (1) comprising an epitope polypeptide H1b of the H1N1 subtype, an epitope polypeptide H3a of the H3N2 subtype, and a nucleic acid adjuvant poly(I:C), wherein the polypeptide H1b, the polypeptide H3a, and the nucleic acid adjuvant poly(I:C) self-assemble to form a composite polypeptide nanocrystal, wherein the polypeptide H1b and the polypeptide H3a are mixed in an equimolar ratio, and the total amount of the composite polypeptide and the poly(I:C) are mixed in a charge ratio of 2; (2) comprising an epitope polypeptide H1b of the H1N1 subtype, an epitope polypeptide H3c of the H3N2 subtype, and a nucleic acid adjuvant poly(I:C), wherein the polypeptide H1b, the polypeptide H3c, and the nucleic acid adjuvant poly(I:C) self-assemble to form a composite polypeptide nanocrystal, wherein the polypeptide H1b and the polypeptide H3c are mixed in an equimolar ratio, and the total amount of the composite polypeptide and the poly(I:C) are mixed in a charge ratio of 2; (3) comprising an epitope polypeptide H1c of the H1N1 subtype, an epitope polypeptide Bc of the IBV subtype, and a nucleic acid adjuvant poly(I:C), wherein the polypeptide H1c, the polypeptide Bc, and the nucleic acid adjuvant poly(I:C) self-assemble to form a composite polypeptide nanocrystal, wherein the polypeptide H1c and the polypeptide Bc are mixed in an equimolar ratio, and the total amount of the composite polypeptide and the poly(I:C) are mixed in a charge ratio of 2; (4) comprising an epitope polypeptide H1a of the H1N1 subtype, an epitope polypeptide Ba of the IBV subtype, an epitope polypeptide Bc of the IBV subtype and a nucleic acid adjuvant poly(I:C), wherein the polypeptide H1a, the polypeptide Ba, the polypeptide Bc and the nucleic acid adjuvant poly(I:C) self-assemble to form a composite polypeptide nanocrystal, wherein the polypeptide H1a, the polypeptide Ba and the polypeptide Bc are mixed in an equimolar ratio, and the total amount of the composite polypeptide and the poly(I:C) are mixed in a charge ratio of 2.

11. The method for preparing a multi-epitope peptide composite vaccine based on the self-assembly principle according to claim 1, characterized in that: The following steps are involved: Dissolving the polypeptide lyophilized powder in a solvent to prepare a polypeptide stock solution; dissolving the nucleic acid adjuvant in a buffer to prepare a nucleic acid adjuvant solution; The polypeptide stock solution and the nucleic acid adjuvant solution are mixed and vortexed to obtain a multi-epitope peptide composite vaccine based on the principle of self-assembly, wherein the composite polypeptide and the nucleic acid adjuvant are mixed at a charge ratio of 2. When a single polypeptide is used, the single polypeptide and the nucleic acid adjuvant form single polypeptide nanocrystals with a periodic microstructure. When two or more composite polypeptides are used, the composite polypeptide and the nucleic acid adjuvant form composite polypeptide nanocrystals with a periodic microstructure. When a single polypeptide or a composite polypeptide and the nucleic acid adjuvant cannot form a periodic microstructure, this type of complex is called a nanocomplex.

12. Use of the multi-epitope peptide composite vaccine based on the self-assembly principle as claimed in claim 1, and use of the multi-epitope peptide composite vaccine based on the self-assembly principle in the preparation of vaccines.

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