Immune carrier microsphere constructed by individualized MHC-II combined polypeptide, vaccine and application of immune carrier microsphere and vaccine

Abstract

The invention relates to the technical field of immune carrier microspheres, in particular to an immune carrier microsphere constructed by individualized MHC-II combined polypeptide, a vaccine and application of the immune carrier microsphere and the vaccine. A core comprises microspheres constructed by a plurality of MHC-II binding polypeptides, sequences of the MHC-II binding polypeptides are obtained based on HLA gene typing results of an inoculator, and each HLA gene corresponds to at least one high-affinity MHC-II binding polypeptide; a shell is a glucan with sulfydryl or other polymer coating layer, and sulfydryl is used as a coupling group to be connected with an antigen to construct the vaccine. The method has the advantages that competition is avoided, inhibition is generated, a better immune effect is achieved, antibodies of high-titer carrier molecules are not generated, and cross reaction side effects are avoided. The carrier molecular diversity is reduced; side effects are avoided. And antibody immunity and cellular immunity functions are generated. The particle size of the microspheres is controllable, and the immunologic function can be achieved without adjuvants. The preparation process is simple to operate and good in repeatability.

Description

Technical Field

[0001] The present invention relates to the technical field of immune carrier microspheres, and in particular to immune carrier microspheres and vaccines constructed with individualized MHC-II binding polypeptides and applications thereof. Background Art

[0002] 1. Research Progress on Therapeutic B Cell Vaccines

[0003] The invention of monoclonal antibody technology in the 1970s was undoubtedly a milestone in medical history, playing an irreplaceable role in disease detection and treatment. Since the beginning of this century, human monoclonal antibody technology has matured, leading to a surge in the launch of therapeutic antibody products, with many more in clinical trials and experimental research, demonstrating that antibodies offer a wider range of therapeutic possibilities.

[0004] B cell vaccines can produce antibodies in the immune system for a long time and have promising applications, but few successful therapeutic products have been marketed. The most researched and developed antibody vaccine in the field of tumor treatment, and the only currently marketed antibody vaccine, is Cuba's CIMAvax-EGF. This vaccine uses epidermal growth factor (EGF) to induce the body to produce EGF antibodies, blocking the binding of EGF to receptors on the surface of cancer cells, thereby inhibiting the growth and proliferation of cancer cells. CIMAvax-EGF provides a new option for adjuvant treatment of non-small cell lung cancer, bringing hope to patients. Its deeper significance lies in that it provides a successful model for the development of therapeutic tumor vaccines, demonstrating the feasibility of fighting cancer by inducing the body to produce specific antibodies, and pointing to hope for subsequent research.

[0005] For benign diseases, there are numerous monoclonal antibodies used for treatment. Therapeutic vaccines, primarily for viral infections, are currently in clinical trials. Fortunately, after nearly 80 years of development, a hypertension therapeutic vaccine has entered Phase III clinical trials and is expected to be commercially available in China and abroad in the next few years. This development represents a potential new treatment option for patients with hypertension and a milestone for therapeutic vaccines.

[0006] Antibodies exert their therapeutic effects in the body in three modes: 1. The products of specific antigen-antibody binding are not excreted directly, but are instead taken up and broken down by phagocytes in the body. This process not only helps clear the antigen-antibody complex but also releases antigenic peptides, further stimulating T cell responses and enhancing immune system function. 2. They prevent functional molecules from binding to receptors, inhibiting their function (EGF vaccine). 3. They bind to antigens on the tumor cell membrane and kill the tumor cells through ADCC (Her2 vaccine).

[0007] 2. Molecular Immunology Basis of B Cell Vaccines and Vectors

[0008] B cells require two activation signals for antibody production. The first signal is the binding of an antigen epitope (B epitope) to the B cell receptor (BCR), a process that determines the specificity of the antibody. The second signal is the release of cytokines by T cells induced by the antigen's Th epitope.

[0009] After binding to the B cell receptor, the exogenous antigen is internalized into the B cell. In the cell, the antigen molecule is hydrolyzed into polypeptide fragments in the lysosome and endosome, and binds to the MHC-II molecules of the B cell, and then displayed on the surface of the B cell. These MHC-II-bound polypeptide epitopes (also called Th epitopes or T epitopes) are presented to CD4 + The cell receptors then release cytokines to activate B cells, which determine their fate, including expansion and antibody production.

[0010] MHC-II binding peptides and Th epitope peptides are the same thing. For B cells or DCs, they are called MHC-II binding peptides, while for T cells, they are called T epitope peptides. Generally speaking, exogenous proteins contain both B epitopes and Th epitopes, and can produce antibodies when immunized in animals and humans.

[0011] Most therapeutic vaccines are prepared from endogenous proteins or peptides. During the development of T cells, T cells with receptors that recognize Th epitopes of endogenous proteins will be cloned and eliminated, and there will be no T cells that recognize endogenous proteins or peptides. For this reason, it is necessary to connect exogenous immune carrier molecules (Immunocarrier) to endogenous antigen molecules to produce antibodies. Traditionally used exogenous molecules are OVA, BSA, KLH, phage or adenovirus, etc., which can provide sufficient diversity of MHC-II binding peptides to adapt to the diversity of animal or human HLA. In recent years, in purebred animal experiments, antigen peptides coupled to single exogenous MHC-II binding peptide molecules as necessary T epitopes have achieved effective results.

[0012] After Th2 cell receptors recognize peptide epitopes bound to exogenous carrier proteins, Th2 cells are activated and proliferate, subsequently releasing cytokines. These cytokines act as a secondary signal for B cell activation, activating B cells to enter a proliferation program and acquire the ability to secrete high-affinity, high-titer antibodies through somatic mutation mechanisms. However, Th cells in a dormant state cannot meet the requirements for B cell activation by themselves. Therefore, dendritic cells (DCs) are required to phagocytose antigens and present Th epitopes to Th cells via MHC-II, thereby activating Th cells.

[0013] It can be seen from this that the secretion of antibodies by B cells is actually a complex process involving two signal molecules: antigen molecules and carrier molecules, and three cells: B cells, Th cells and DC cells.

[0014] DCs tend to phagocytose particulate antigens, so soluble proteins are generally difficult to produce high-titer antibodies. Traditional methods involve formulating soluble proteins into emulsion particles or adsorbing them onto immune adjuvant particles to achieve higher antibody titers. However, these methods primarily produce antibodies targeting highly immunogenic epitopes on the exogenous carrier molecule, resulting in immune competitive inhibition of the target antigen and very low antibody titers. Using MHC-II microspheres as carriers allows for the production of high-titer antibodies without an immune adjuvant.

[0015] III. Risks of Therapeutic Vaccines and Countermeasures

[0016] 1. Associations with the side effects of the new crown vaccine

[0017] COVID-19 vaccines produce antibodies against the S protein of the Covid-19 virus, blocking the virus from binding to the ACE2 receptor and preventing infection. In terms of antibody titers, mRNA vaccines produce the highest titers, ranging from hundreds to thousands. Adenovirus-expressing vaccines have titers ranging from hundreds to over a thousand, and inactivated virus vaccines have titers in the hundreds. Literature reports suggest that COVID-19 vaccines have an alarmingly high incidence of side effects. While traditional vaccines only cause localized symptoms at the injection site, COVID-19 vaccines can cause systemic symptoms, and a small number of people experience long-term side effects in the immune, nervous, and cardiovascular systems.

[0018] The most common side effects reported in the literature include fatigue, headache, myalgia, fever, pain or redness at the injection site, accompanied by mild or moderate symptoms. On the other hand, some rare adverse events have also been reported, such as allergic reactions after mRNA vaccines, and thrombosis and thrombocytopenia after vaccination with non-replicating viral vector vaccines. Other rare adverse events described include myocarditis, Bell's palsy, transient myelitis, Guillen-Barre syndrome, herpes zoster recurrence, autoimmune attacks, epilepsy and orthostatic tachycardia.

[0019] The reason for this, as one might suspect, is that the high dose required to produce high antibody titers. The vaccine's poor preventive efficacy and significant side effects surprised researchers. Therapeutic vaccines are required to produce higher antibody titers than preventative vaccines, and if they exhibited similar side effects to the COVID-19 vaccine, it would likely fail Phase III clinical trials and be marketed.

[0020] 2. Analysis of the sources of vaccine side effects

[0021] (1) B cells produce antibodies to ineffective components of the vaccine: Taking the new coronavirus vaccine as an example, only antibodies produced against the S protein and ACE2 receptor binding site can effectively block viral transmission. Antibodies produced against other components of the viral coat protein are not only ineffective, but can also produce cross-reaction side effects. Inactivated virus vaccines and adenovirus vaccines will produce high-titer membrane protein antibodies, and even S protein vaccines will produce antibodies to irrelevant target epitopes. The specificity of antibodies is relative. Irrelevant polyclonal antibodies will cross-react with normal tissue molecules, and the ADCC effect will kill irrelevant cells and tissue organs. There are even antibody-mediated infections of cells without ACE2 receptors, namely the ADE effect.

[0022] In current research on preparing vaccines using peptides as antigens, in most cases coupling the peptides to KLH carrier molecules can achieve good immune effects, but the antibody titer produced against KLH will be much higher than that of the antigen molecule, and the side effects of antibody cross-reactions cannot be determined.

[0023] (2) Overactivation of T cells: In addition to irrelevant antibodies, DC cells can present vaccine carrier molecular components to activate T cells, produce excessive cytokines, activate CD8 cytotoxic T cells to damage normal tissues, and produce side effects.

[0024] For example, KLH (KLH) is considered a safe immune carrier molecule for clinical use. Immunization can cause excessive cytokine secretion by T cells, leading to systemic side effects. In fact, KLH is an ideal antigen for inducing DTH. Keyhole limpethemocyanin (KLH) is a highly immunogenic protein macromolecule that enables dendritic cells to recognize Th1 cells. It is an ideal antigen for inducing late DTH. KLH is injected into mice and then locally reinjected several weeks later. The DTH response is assessed by relative swelling at the injection site.

[0025] After KLH immunization, IL-6 secretion increased significantly, ranging from 4 to 16 times. IL-6 is an important inflammatory factor, and its increase is generally associated with the activation of the immune response. IL-6 can lead to the development of chronic inflammatory diseases (such as rheumatoid arthritis) and cytokine storms, which occur in patients treated with CAR-T therapy and in various diseases, including those infected with the new coronavirus SARS-CoV-2.

[0026] After KLH immunization, the secretion of IFN-γ increased by 21 times, ranging from 5 to 45 times. IFN-γ is an important immune regulatory factor. Possible side effects of interferon include pain at the injection site, inflammation, fever, chills, headache, myalgia, joint pain, fatigue, dizziness, anorexia, diarrhea, weight loss, leukopenia in blood count, thrombocytopenia, mild hair loss, oral ulcers, transient rash, delusions, depression, decreased concentration, insomnia, anxiety, irritability, autoimmune diseases such as autoantibodies, thyroid disease, diabetes, blurred vision, dry eyes, eye inflammation, hearing loss, menstrual disorders, loss of libido, pharyngitis, rhinitis, etc.

[0027] After KLH immunization, the secretion of TNF-α increased significantly, ranging from 5 to 50 times. TNF-α is an important inflammatory factor associated with various inflammatory reactions.

[0028] C-reactive protein (CRP): Although the relationship between KLH and elevated CRP is not directly mentioned, elevated IL-6 levels often induce CRP production, so KLH-induced increases in IL-6 may indirectly lead to elevated CRP levels. These cytokines and inflammatory molecules can cause systemic reactions such as fever, headache, muscle aches, joint pain, gastrointestinal discomfort, and allergic reactions. Involved cytokines include IL-4, IL-5, and IL-13, and measurable chemicals include histamine, tryptase, leukotrienes, and prostaglandin D.

[0029] Attenuated vaccines, adenovirus vector vaccines (VLP vaccines) theoretically produce similar results to KLH.

[0030] The antigens expressed by mRNA vaccines are relatively simple, but the expression system products are very complex. According to literature reports, the side effects of mRNA vaccines may come from the liposome packaging materials of mRNA.

[0031] 3. Corresponding measures

[0032] (1) Avoiding the production of high-titer antibodies to the carrier molecule: Cross-reactions may produce side effects. The immune carrier molecule is placed inside the microsphere. Two patents have been applied for: immune microspheres that overcome B cell immune tolerance and their applications (application number: CN201010571277.1); fully diverse amphiphilic MHC II binding peptides, immune carrier microspheres and their preparation methods and applications (application number: CN201910115133.6). This patented microsphere technology is a process improvement.

[0033] (2) Avoid using large exogenous molecules as immune vectors

[0034] To address the diversity of HLA in the human population, traditional carrier molecules include macromolecules such as BSA, OVA, KLH, viruses, and a wide variety of random peptides. The present invention selects personalized, specific MHC-II-binding peptides to avoid systemic side effects caused by overactivation of T cells.

[0035] 4. Feasibility analysis

[0036] Individualized MHC-I binding peptides for antigen epitopes are used in tumor cell vaccine therapy. HLA assay technology is mature, widely available, and affordable. HLA-2 has three genetic alleles: HLA-DR, HLA-DP, and HLA-DQ. Allele mutations and inheritance vary by ethnicity and region. Globally, there are thousands of HLA-2 alleles, but the diversity and frequency of these alleles in the Han Chinese population are as follows:

[0037] HLA-DR:

[0038] HLA-DRB1*09:01: approximately 15%-25%

[0039] HLA-DRB1*12:02: approximately 10%-20%

[0040] HLA-DRB1*15:01: approximately 5%-15%

[0041] HLA-DRB1*04:05: approximately 5%-10%

[0042] HLA-DRB1*07:0: approximately 5%-10%

[0043] HLA-DRB1*08:03: approximately 3%-8%

[0044] HLA-DRB1*11:01: approximately 3%-8%

[0045] HLA-DRB1*14:05: approximately 2%-5%

[0046] HLA-DRB1*01:01≤5%

[0047] HLA-DRB1*03:01≤5%

[0048] HLA-DRB1*13:01≤5% or above 11 types account for more than 95% of the population.

[0049] HLA-DPB1 is highly diverse, and the common subtypes and their proportions are roughly as follows:

[0050] HLA-DPB1*05:01: approximately 20%-30%

[0051] HLA-DPB1*02:01: approximately 15%-25%

[0052] HLA-DPB1*04:01: approximately 10%-20%

[0053] HLA-DPB1*03:01: approximately 5%-10%

[0054] HLA-DPB1*09:01: approximately 5%-10%

[0055] Other subtypes (such as HLA-DPB1*13:01, HLA-DPB1*14:01, etc.): The proportion is usually less than 5%. The abundance ratio of HLA-DQB1 subtypes is higher. The diversity of HLA-DQB1 genes is higher. The common subtypes and their abundance ratios in the Chinese Han population are as follows:

[0056] High-frequency subtype:

[0057] HLA-DQB1*03:01: approximately 20%-30%

[0058] HLA-DQB1*03:03: approximately 15%-25%

[0059] HLA-DQB1*06:01: approximately 10%-20%

[0060] IF subtype:

[0061] HLA-DQB1*02:01: approximately 5%-10%

[0062] HLA-DQB1*05:01: approximately 5%-10%

[0063] HLA-DQB1*06:02: approximately 5%-10%

[0064] Individualized carrier microspheres do not require thousands of designs; only 30-50 designs are sufficient to meet the needs of over 90% of the population. MHC-2 binding peptides can be designed using online software, with similar results obtained using different software. Peptide synthesis technology is mature and inexpensive. The technology presented in this paper is easy to use and highly reproducible, and the microsphere preparation method is simple and repeatable.

[0065] MHC-2 binding peptide prediction: The annual increase in HLA gene diversity is a result of sequencing. HLA-2 gene mutations and corresponding peptide sequences are well-known, and years of experience are accumulated in predicting binding peptide sequences. Various sequence prediction software programs are available, including IEDB ProPred16, MHCPred17, SVRMHC18, ARB19, SMM-align20, and NetMHCIIpan. IEDB and NetMHCPan are relatively easy to use. Prediction results for HLA-DR are generally consistent, but for HLA-DQ and HLA-DP, there are significant discrepancies. NetMHpanC4.3, which has been upgraded several times, is more reliable and has been widely cited.

[0066] In addition, computer-designed peptides such as AKFVAAWTLKAAA, TT830–844ILMQYIKANSKFIGI, and TT947-967 diphtheria toxin QSIAISSLMAQAIP can bind to multiple MHC-2 molecules, and this universal Th epitope can serve as a supplement. Summary of the Invention

[0067] The purpose of the present invention is to overcome immune competitive inhibition, increase antibody titers, avoid the side effects of irrelevant antibody cross-reactions, and avoid the side effects of T cell overactivation. In order to adapt to human HLA diversity, personalized MHC-II binding polypeptide microspheres are used to replace immune carrier macromolecules containing sufficient diversity MHC-II: OVA, BSA, KLH, phage, virus or single MHC-II molecules.

[0068] To achieve the above objectives, a core-shell structured immune carrier microsphere constructed with personalized MHC-II binding polypeptides is provided. The microsphere comprises a core and an outer shell; the core comprises microspheres constructed with several individually designed MHC-II binding polypeptides, the sequences of which are obtained through computer prediction and screening based on the HLA genotyping results of the vaccine recipient, and each HLA allele corresponds to at least one high-affinity MHC-II binding polypeptide; the outer shell is a thiol-containing dextran or other polymer coating layer, the thiol groups of which serve as coupling groups for connecting antigens to construct a vaccine.

[0069] Preferably, the immune carrier microspheres contain 3-12 computer-screened high-affinity MHC-II binding polypeptides and at least one universal MHC-II binding polypeptide for assisting B cells in producing antibodies, wherein the universal MHC-II binding polypeptide is selected from at least one of the following:

[0070] YFAVYLQETPY,

[0071] AKFVAAWTLKAAA,

[0072] QYIKANSKFIGITEL,

[0073] FNNFTVSFWLRVPKVSASHLE,

[0074] AWLEAQEEEEVGF,

[0075] QYIKANSKFIGITELKK,

[0076] EPRAPWIEQEGPEYWDQE,

[0077] ADVEVYRAVTPLGPPD,

[0078] DTLRSYYADWYQQKPG,

[0079] FVNQHLAGSHLVEAL,

[0080] LNEDLRSWTAADTAA.

[0081] Preferably, the MHC-II binding polypeptide is obtained by affinity scoring screening based on HLA typing using IEDB ProPred, MHCPred, SVRMHC, ARB, SMM-align, NetMHCIIpan, IEDB and NetMHCPan prediction software.

[0082] Preferably, the particle size of the microspheres is 0.1-10 microns, and the microspheres are one of self-assembled MHC-II binding polypeptide self-assembled microspheres, MHC-II binding polypeptide modified PLGA microspheres or albumin microspheres containing MHC-II binding polypeptides. The particle size of the MHC-Ⅱ binding polypeptide self-assembled microspheres is 2000-5000 nm, the particle size of the MHC-Ⅱ binding polypeptide coupled PLGA-PEG microspheres is 500-1000 nm, and the particle size of the HSA microspheres containing MHC-Ⅱ binding polypeptides is 1-10 μm. The particle size of the microspheres is controllable to facilitate DC phagocytosis.

[0083] The present invention also provides a method for preparing immune carrier microspheres, comprising the following steps:

[0084] S1: Individualized MHC-II binding peptide design: Based on the individual's HLA gene test results, computer software is used to screen high-affinity MHC-II binding peptide sequences;

[0085] S2: Preparation of microsphere core: The screened peptide sequence is synthesized in a professional company, and protein microspheres containing MHC-binding peptides are prepared as cores through self-assembly, peptide-modified PLGA microspheres or emulsification methods;

[0086] S3: Shell coating: The core of the microsphere is coated with oxidized dextran with thiol or other polymer materials, which react with the core microspheres to form core-shell structure immune carrier microspheres. Other polymer materials include at least glucomannan.

[0087] The present invention also provides a vaccine prepared based on immune carrier microspheres, wherein the individualized MHC-II binding polypeptide is packaged in the microspheres as an immune carrier molecule, and the antigen molecules of the vaccine are coupled to the surface of the microspheres to form a B cell microsphere vaccine.

[0088] Preferably, the microspheres replace KLH, phage, and viral immune carrier molecules for the preparation of new vaccines using endogenous proteins or polypeptides. The B cell microsphere vaccine constructed with MHC-II binding polypeptide microspheres produces better immune effects than the commonly used KLH immune carrier vaccine, and the antibody titer produced against the MHC-II polypeptide carrier molecule is much lower than that of the commonly used KLH.

[0089] Preferably, the microspheres are loaded with MHC-II and T cell epitope polypeptides, and are used to prepare T and B mixed vaccines with antibody immunity and cellular immunity effects.

[0090] Preferably, the surface of the microspheres is coupled with a variety of different antigens to construct a new vaccine.

[0091] The present invention also relates to the use of therapeutic vaccine preparations, wherein the vaccine is used in the preparation of drugs for tumors, cardiovascular diseases, hyperglycemia, allergic diseases, autoimmune diseases, Alzheimer's disease, viral infections, drug addiction treatment, weight loss and other chronic diseases that can be treated with antibodies.

[0092] Compared with the prior art, the present invention has the following advantages:

[0093] 1. Vaccines prepared with carrier microspheres produce high antibody titers upon vaccination. The present invention is based on the separation of T and B cell epitopes inside and outside the microspheres, which avoids competition and inhibition between exogenous carrier molecules and antigen epitopes.

[0094] 2. The carrier molecules are packaged in microspheres, which will not produce high-titer antibodies against exogenous carrier molecules, thus avoiding side effects caused by antibody cross-reactions.

[0095] 3. Reduce the diversity of MHC-II binding peptides to avoid over-activation of T cells and side effects.

[0096] 4. In addition to packaging MHC-II binding peptides, microspheres can also be loaded with MHC-I binding peptides, i.e., T cell antigen peptides. Vaccines prepared in this way can generate both antibody immunity and cellular immunity. Similarly, multiple antigen peptides can be linked to the surface of the microspheres.

[0097] 5. The particle size of microspheres is controllable and can exert immune function without adjuvants, which is more suitable for vaccines for benign diseases.

[0098] 6. The preparation process of the present invention is simple to operate, the vaccine structure is controllable, and the repeatability is good. DETAILED DESCRIPTION

[0099] The present invention is further described below with reference to examples, which are only used for explanation and are not intended to limit the scope of protection of the present invention.

[0100] Example 1 Design of Personalized MHC-II Binding Peptides (i.e., Th2 Epitope Peptides)

[0101] To design personalized MHC-II binding peptides, the vaccine recipient first uses the PSS-PCR method to determine the HLA-II allele type subtype.

[0102] There are several computer applications available online for predicting MHC-binding peptides: IEDB ProPred16, MHCPred17, SVRMHC18, ARB19, SMM-align20, and NetMHCIIpan. IEDB and NetMHCPan are relatively easy to use. Their prediction results for HLA-DR are generally consistent, but they differ significantly for HLA-DQ and HLA-DP. NetMHpanC4.3, which has undergone numerous upgrades, is widely cited in the literature, and more advanced applications are expected in the future.

[0103] According to the gene name number of HLA-II typing determination, the foreign protein sequence used for prediction is pasted into the table for calculation by computer software to output the high-affinity MHC-II binding 15 peptides.

[0104] Chicken ovalbumin, bovine serum albumin, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, HIV virus, mazhen virus, tetanus toxin, KLH are usually used, and the 15-peptide sequences with high scores (high affinity) are selected in the result column.

[0105] (1) Taking HLA-DRB10901 as an example, the following results are obtained in Table 1:

[0106] Table 1

[0107] Protein source Export of MHC-II binding peptides Affinity score HcV GPSYGFQYSPAERVD 0.9621 KLH EDVFHPSEPFFIKVS 0.9563 HaV MRNEFRVSTTENVVN 0.9393

[0108] (2) Taking HLA-DQA1*01:01 / HLA-DQB1*05:01 as an example, the following results in Table 2 were obtained using NetMHCPan software:

[0109] Table 2

[0110]

[0111]

[0112] (3) Using NetMHCPan software with DPA1*01:03 / DPB1*04:01, the following results are obtained in Table 3:

[0113] Table 3

[0114] Protein source Export of MHC-II binding peptides Affinity score HcV PDKEILYEAFDEMEE 0.97 KLH NEAIFQQTKFGEFSS 0.98 TT LYTSYLSITFLRDFW 0.84

[0115] A high score indicates a high affinity, and a score of 0.9 or above indicates an affinity at the nM and pM levels.

[0116] The mice used in the experiment are purebred animals with a small number of H2 alleles. The H2 gene polymorphism is as follows:

[0117] The H2 alleles of Balb / c mice are H2-Kd, H-Ld, and H2-Dd in class I and I-Ed and I-Ad in class II.

[0118] Using OVA, TT, and KLH sequences, the following H2I-Ed and H2I-Ad binding peptides can be obtained:

[0119] Table 4: Balb / c mice

[0120] H2I-Ad OVA LKISQAVHAAHAEIN 0.8638 H2I-Ad TT YDTEYYLIPVASSSK 0.7569 H2I-Ad TT GTNEYSIISSMKKHS 0.2785 H2I-Ad KLH NIDFLDKKTNRAVDD 0.3590

[0121] The H2 alleles for C57BL / 6 mice are H2-Kd and H2-Kb for class I and I-Ab for class II:

[0122] Table 5: C57BL / 6 mice

[0123] H2-IAb TT VPYIGPALNIVKQG 0.9531 H2-IAb TT TEYYLIPVASSSKDV 0.8934 H2-IAb KLH DDEYAGSFVILGGAK 0.8686

[0124] Example 2 Preparation of Oxidized Dextran

[0125] The purpose of preparing oxidized dextran is to oxidize the hydroxyl groups of dextran to generate aldehyde groups, which react with amino groups on the microspheres to form coated core microspheres. The excess aldehyde groups then react with mercaptopropylamine to introduce sulfhydryl groups onto the dextran molecules. This is used to link antigens to prepare peptide vaccines. Dextran oxidation (aldehyde formation) introduces aldehyde groups (-CHO) by oxidizing the vicinal diol structure of dextran, providing active sites for subsequent coupling reactions.

[0126] Materials: Dextran T40, sodium periodate (NaIO4, 0.1M), PBS buffer (pH 6.0-7.0), dialysis bag (molecular weight cut-off is selected according to dextran)

[0127] step:

[0128] (1) Dissolving dextran: Dissolve dextran in PBS buffer (pH 6.0-7.0) to prepare a 5-10 mg / mL solution.

[0129] (2) Oxidation reaction: Add NaIO4 to a final concentration of 0.05-0.1 M (protect from light and use an ice bath) and stir at room temperature for 4-6 hours (or at 4°C overnight).

[0130] (3) Termination of the reaction: Ethylene glycol (final concentration 0.1 M) was added to quench the excess NaIO4 and stirring was continued for 30 minutes.

[0131] (4) Purification: The solution was transferred to a dialysis bag and dialyzed against PBS (pH 7.4) for 24 hours (changing the solution three times) to remove unreacted reagents. The oxidized dextran (labeled as Dex-CHO) was freeze-dried and stored.

[0132] (5) Since DC cells have mannose receptors, a certain proportion of glucomannan was further added to the glucan.

[0133] Example 3 Preparation of three types of microspheres with MHC-II binding polypeptides

[0134] 1. Preparation of MHC-Ⅱ binding peptide self-assembled microspheres

[0135] The self-assembling peptide method is roughly the same as that in Patent 201810151476.3. The difference is that in the aforementioned patent, MHC-II binding peptides and antigen peptides are linked together to prepare self-assembling microspheres, while the present invention is limited to MHC-II binding peptides (15 peptides), which are easy to design and self-assemble, and then coated with dextran to stabilize them and be used to attach antigens.

[0136] The hydrophilic portion of the self-assembling amphiphilic peptide is designed to be insoluble in water but soluble in water / acetonitrile. The hydrophobic ends of two different peptides adhere to each other through hydrophobic binding forces, while the amino acids in the hydrophilic portions attract each other with similar positive and negative ionic charges, forming self-assembling peptide microspheres.

[0137] Self-assembly: Dissolve the self-assembling peptide in a mixed solvent of water and acetonitrile (volume ratio 1:1) at a concentration of 1 mg / ml, and remove the acetonitrile by rotary evaporation.

[0138] During the volatilization of acetonitrile, the self-assembling peptide gradually self-aggregates into spheres, and finally an aqueous solution containing self-assembling peptide microspheres is obtained. The self-assembling peptide microspheres are obtained by freeze-drying, and the particle size of the microspheres is about 2-7 microns.

[0139] 2. Preparation of PLGA-PEG microspheres linked to MHC-Ⅱ binding peptides

[0140] The technology for thiol-containing PLGA-PE-SH microspheres is highly mature. Microspheres with a designed particle size of 500 nm were commissioned to a company for preparation. The MHC-binding peptide with an N-terminal maleimide group was commissioned to a peptide synthesis company. The company also linked the peptide to the microspheres. It is estimated that each microsphere contains over 100 peptides.

[0141] 3. Preparation of MHC-II binding peptide-human serum albumin immune carrier microspheres

[0142] (1) The concentration of human serum albumin with an appropriate amount of thiol groups introduced is 5-10%, and adjuvant molecules such as MHC-2 binding peptides, intracellular immune adjuvant CpG nucleotides, and cytokines are added. The serum albumin microspheres have a particle size of 3-5 μm and are manufactured by a microfluidics technology company.

[0143] (2) Under microfluidic conditions, an ethanol-isoamyl alcohol organic phase is added dropwise. Ethanol is added under stirring to absorb water and precipitate to form microspheres with a particle size of 3-10 microns.

[0144] (3) The microspheres are added to a pH 8.5 tris buffer solution. Under oxidative conditions, the albumin is solidified through disulfide bonds. The other components remain in a free and active state.

[0145] Example 4 Oxidized dextran coated MHC binding polypeptide microspheres

[0146] Objective: To covalently bind oxidized dextran to the surface of MHC-binding peptide microspheres by utilizing the reaction of aldehyde groups with amino groups of proteins or peptides.

[0147] Materials and reagents: oxidized dextran, microspheres, sodium cyanoborohydride (NaBH3CN, 0.1 M), PBS buffer (pH 7.4)

[0148] step:

[0149] (1) Activation of microspheres: The protein microspheres were suspended in pH 7.8 phosphate buffer (concentration 1-5 mg / mL).

[0150] (2) Coupling reaction: Add Dex-CHO solution (final concentration 1-2 mg / mL) and stir at room temperature for 4-6 hours; add NaBH3CN (final concentration 5-10 mM) and carry out reduction reaction at 4°C for 2 hours to stabilize the Schiff base.

[0151] (3) Purification: Centrifuge (10,000 rpm, 10 minutes) to remove unbound Dex-CHO; wash three times with pH 7.4 PBS to obtain Dex-CHO-coated protein microspheres.

[0152] Example 5 Introduction of sulfhydryl groups into oxidized dextran and coupling of antigenic polypeptides

[0153] Objective: To introduce sulfhydryl groups (-SH) by reacting the residual aldehyde groups of oxidized dextran with the amino groups of mercaptopropylamine.

[0154] step:

[0155] (1) Oxidized dextran microspheres were suspended in carbonate buffer (pH 8.5) to enhance the amino-reactivity.

[0156] (2) Add mercaptopropylamine (final concentration 5-10 mM) and stir at room temperature for 6-8 hours. Add NaBH3CN (final concentration 5 mM) and allow to reduce at 4°C for 2 hours to stabilize the connection. Wash three times by centrifugation (PBS, pH 7.4) to remove unreacted mercaptopropylamine. The final product is Dex@Protein-SH microspheres.

[0157] (3) Thiol quantification and blocking: Ellman's reagent is used to detect the content of free thiol (-SH) groups on the surface of microspheres. At the same time, the disulfide bonds are blocked and the nitrobenzoic acid surface of the microspheres is negatively charged, which makes the microspheres dispersible. The number of thiol groups on the surface of the microspheres is controlled to be 300-1000.

[0158] (4) Antigen coupling: The microspheres are suspended in PBS, and mercaptoethanol or mercaptothreitol is added to release the closed disulfide bonds for antigen coupling. After one minute, the 4-mercapto-2-nitrobenzoic acid is centrifuged to remove the dissociated 4-mercapto-2-nitrobenzoic acid. When synthesizing polypeptide antigens, a maleimide group is introduced at the N-terminus. Protein antigens are reacted with N-hydroxysuccinylmaleimide ester reagent to introduce a maleimide group. The antigen is coupled to the microspheres in the presence of EDTA and stirring, with the reaction solution filled with nitrogen to remove oxygen. The reaction is then placed in a refrigerator at 4 degrees overnight. The unreacted thiol groups on the microspheres are blocked with iodoacetic acid.

[0159] (5) Centrifuge to remove unattached antigens.

[0160] (6) Turbidity is used to determine the concentration of microspheres, and OD280 is used to determine the approximate protein content.

[0161] Example 6 Preparation of anti-IgE polypeptide vaccine using MHC-II binding polypeptide self-assembly microspheres

[0162] Allergens bind to IgE antibodies, causing the Fc of immunoglobulin IgE to bind to the high-affinity receptor FcεRI on the surface of effector cells, causing rapid degranulation of mast cells and basophils, releasing stored histamine, and then synthesizing and releasing prostaglandins, leukotrienes, cytokines, and other inflammatory mediators. These attract and activate inflammatory cells, produce allergic symptoms, and upregulate the biosynthesis of IgE by B cells to promote increased sensitivity.

[0163] Omazumab is currently the world's first approved humanized anti-IgE monoclonal antibody, an antibody drug used to treat asthma, blocking the binding of IgE to its receptor. Monoclonal antibodies targeting IgE-Fc receptor II (FcεR1) are currently under development.

[0164] Research is underway in various countries on developing vaccines using IgE and IgE receptors as antigens to generate endogenous antibodies. It is expected that combining the two will be even more effective.

[0165] The animal experiment used mouse IgE peptide as antigen, the sequence is: GYGYQCIVDHPDFP (Master's thesis: Receptor binding site multimeric antigen peptide inhibits allergic asthma in mice).

[0166] The mouse IgE antigen peptide GYGYQCIVDHPDFP was commissioned to be synthesized by a company with a maleimide-based peptide at the N-terminus.

[0167] The MHC-II binding peptide LKISQAVHAAHAEIN corresponding to the H2 gene of Balb / c mice is a self-assembling microsphere peptide, and the self-assembling peptide microspheres with thiol groups are prepared according to the above method.

[0168] The IgE antigen polypeptide was coupled to the microspheres and used as a vaccine for experimental research. Balb / c mice were immunized four times. Six weeks later, blood was collected to measure the antibody titer (see Table 6).

[0169] Table 6 IgE antigen peptide and self-assembled MHC-II peptide antibody titer

[0170]

[0171]

[0172] The synthesis of antigen peptides, coupled to KLH as experimental vaccines for mice and the determination of antibody titers are routine technical services provided by Peptide Synthesis Company. The immune results of the conjugate of IgE peptides linked to KLH are shown in Table 7:

[0173] Table 7 Results of antibody titers in mice vaccinated with IgE-KLH polypeptide vaccines

[0174]

[0175] Results from two experiments showed that both IgE peptide antigens linked to the novel immune carrier microspheres and KLH produced high antibody titers. The microsphere vaccine was more effective than the KLH protein vaccine, with titers approximately four times higher. A more significant difference was that the antibody titers for the carrier molecule KLH and the MHC-II-binding peptide differed by over 100 times.

[0176] Several antigenic peptide epitopes have been identified in the extracellular region of the high-affinity IgE receptor FcεRI on eosinophils and basophils. Several linear peptide epitopes, including QCRVTHPHLPRALMRS and ADSNPRGVSAYLSRPSPGGC, are located in the Cε3 region of the Fc region of human IgE and can serve as antigens to generate antibodies that effectively block IgE binding to the receptor. Combining these two may be more effective.

[0177] Example 7: Preparation of Cardiovascular and Cerebrovascular Disease Therapeutic Vaccines Using MHC-II Binding Peptide-Modified PLGA Microspheres

[0178] PLGA microspheres are one of the few microspheres approved for clinical use by the FDA. The preparation process is mature and the particle size is controllable. Microspheres made of PLGA-PEG-SH contain thiol groups for coupling MHC-binding peptides and are suitable for therapeutic vaccines for benign diseases.

[0179] Cardiovascular and cerebrovascular diseases, including coronary heart disease, hypertension, myocardial infarction, and stroke, are the leading causes of death worldwide. They are caused by the accumulation of oxidized low-density lipoprotein (LDL) particles, which lead to the accumulation of atherosclerotic plaques in arteries.

[0180] 1. Lipid-lowering vaccines

[0181] Lipids are important energy, structural, and hormonal components of the human body. Fat is insoluble in water and plasma and must be bound to apolipoproteins to be transported throughout the body. There are three antibody vaccine targets for lowering blood lipids:

[0182] The first target is apolipoprotein B-100, the main component of LDL. Two linear epitope peptides, p210 and p45, of ApoB-100 are used as immunogens. The Austrian company AFFiRiS has developed two peptide vaccines that are now in clinical trials. Phase IIa results have shown they can lower LDL levels.

[0183] The second target is cholesteryl ester transfer protein (CETP). CETP is a plasma glycoprotein secreted by the liver. Its primary function is to mediate the bidirectional transfer of cholesteryl esters and triglycerides from high-density lipoprotein (HDL) to low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL). Inhibiting CETP activity helps increase HDL levels and lower LDL and VLDL levels, potentially reducing the risk of atherosclerotic cardiovascular disease.

[0184] In 2004, AVANT, a US company, announced the results of a Phase 1 clinical trial of a CETi vaccine specifically designed to stimulate the production of CETP antibodies. The study enrolled 203 patients with low HDL-C levels, two-thirds of whom were not receiving statins. These patients received either a placebo or the CETi vaccine. Six months later, the vaccine group showed an 8.4% increase in HDL-C levels compared to baseline, particularly in those receiving the high-dose vaccine. This is a promising result. The human CETP antigen peptide sequence is referenced in the literature as (aa461–476): FGFPEHLLVDFLQSLS).

[0185] The third vaccine target molecule is PCSK9. PCSK9 is a secreted protease expressed in the liver that regulates the blood concentration of low-density lipoprotein cholesterol (LDL-C) and is positively correlated with LDL-C. PCSK9 antibodies inhibit PCSK9 function, increasing the number of LDLRs and thus significantly lowering LDL-C levels.

[0186] Currently, there are three PCSK9 monoclonal antibodies on the market: evolocumab, alirocumab, and tolcizumab. There is also an siRNA on the market that inhibits PCSK9 expression and lowers blood LDL.

[0187] There are several human PCSK9 epitope peptides, one of which is (aa207–223): NVPEEDGTRFHRQASKC). We commissioned a specialized company to synthesize an N-terminal maleimide-containing peptide. This peptide was then coupled to PLGA immune microspheres modified with an MHC-II binding peptide to prepare a pilot vaccine. The commissioned company completed the mouse animal testing, immunizing three times and measuring antibody titers five weeks later. The results are shown in Tables 8 and 9:

[0188] Table 8 Antibody titers in Balb / c mice immunized with PCSK9 peptide-microsphere vaccines

[0189]

[0190]

[0191] Table 9 PCSK9 polypeptide-KLH conjugate immunization Balb / c mouse antibody determination results

[0192]

[0193] The results showed that, similar to IgE vaccines, MHC-II-bound peptide immune microspheres produced higher titers of PCSK9 peptide antibodies than KLH carrier molecules, but KLH molecules produced higher titers of antibodies. Antibodies to MHC-II-bound peptide molecules were approximately 100-fold lower than those to KLH.

[0194] 2. Hypertension Vaccine

[0195] Hypertension is a symptom of a disease primarily caused by vascular problems: atherosclerosis, stenosis caused by plaque buildup, and neuroendocrine dysfunction. Hypertension is the leading cause of death worldwide. According to the World Health Organization (WHO) in 2023, only one-fifth of the world's 1.3 billion patients have blood pressure within the recommended range.

[0196] Hypertension therapeutic vaccines are an emerging immunomodulatory strategy that aims to achieve long-term blood pressure control by inducing antibodies to neutralize endogenous pressor factors.

[0197] The target molecules of the hypertension treatment vaccine are angiotensin II and its precursor molecules, as well as the angiotensin II receptor molecules. Currently, a series of sartan drugs are clinically available to inhibit the binding of Ang II to the receptor, with the side effect of damaging the liver and kidneys.

[0198] The vaccine currently in clinical trials is CYT006-AngQb, developed by Swiss company Cytos. It directly couples Ang II (octapeptide: DRVYIHPF) to the carrier protein KLH, and has demonstrated efficacy in clinical trials. Another vaccine currently in clinical trials is ATRQβ-001, a virally expressed Ang II receptor vaccine. ATRQβ-001 has demonstrated antihypertensive effects in Phase II trials. It is constructed using the Qβ phage-expressed AT1R heptapeptide fragment, CAFHYESQ.

[0199] This invention proposes coupling the AngII 8 peptide and the extracellular 7-peptide fragment of the AT1R receptor to the same integrated MHC-II binding peptide-PLGA immune carrier microspheres, which is expected to have better efficacy and can be approved as a single drug. Preliminary results from pilot studies using mice are shown in Table 10:

[0200] Table 10 Immunization results of angiotensin (octapeptide) and angiotensin receptor polypeptide fragment (septapeptide) microsphere vaccine

[0201]

[0202] The results showed that the two peptides were antigenically similar and could potentially be used as a therapeutic vaccine.

[0203] Example 8 Preparation of Anti-tumor Vaccine Using MHC-II Binding Polypeptide Protein Microspheres

[0204] Cancer is a difficult disease to treat, as traditional chemotherapy drugs are highly toxic and prone to drug resistance. Ideally, therapeutic vaccines for cancer are a safe and effective treatment option, and represent the most researched area of ​​therapeutic vaccine product development. While safety is paramount for vaccines used to treat benign diseases, efficacy is crucial for cancer vaccines.

[0205] Tumor vaccines using EGF as an antigen have shown efficacy in some non-small cell lung cancers. This study used commercially available mouse EGF protein coupled to protein microspheres containing two mouse MHC-II binding peptides and constructed with mouse serum proteins. Five C57 mice were immunized, and after five weeks, antibody titers averaged 40,000, though this did not confirm superior clinical efficacy. This is primarily due to the high heterogeneity of human tumor cells, which vary from person to person and from cell to cell, resulting in varying antigens, immunogenicity, and HLA levels. In particular, in the past, single-target vaccines were used in clinical trials, consistently producing positive results in animal models. Phase I trials were safe, Phase II trials were effective, and Phase III trials failed. However, only melanoma antigens are well-researched, and combined multi-target vaccines have demonstrated promising results.

[0206] Tumor treatment vaccines using multiple targets and the combined use of B cell antigens and T cell antigens are expected to improve treatment outcomes.

[0207] Tumor therapy vaccines involve three types of personalized molecules: MHC-I binding peptides (T cell antigens), MHC-II binding peptides, and B cell antigens. Each molecule is diverse, making it more suitable to prepare vaccines using protein immune carrier microspheres. The diverse MHC binding peptides do not need to be coupled and loaded into the microspheres; only the B cell antigens are attached to the microsphere surface.

[0208] The effectiveness of tumor treatment vaccines may depend on antigen detection, and the detected antigens should be loaded into microspheres as much as possible. Taking lung cancer as an example, the lung cancer cell membrane has the following antigens as shown in Table 11:

[0209] Table 11

[0210]

[0211] The above are some materials of commonly used target molecules for immunotherapy of lung cancer. In addition, there are EGF, CEA, MUC1, WT1, P53 mutants, KRAS, and other molecular T and B cell antigen molecular epitopes, as well as individualized point mutations, splicing shifts and other antigens, which need to be fully measured to obtain results.

[0212] Tumor therapeutic vaccines involve complex tumor molecular biology and tumor immunology, but from a technical perspective, the above-mentioned multiple lung cancer B cell antigen peptides can be connected to the surface of microspheres, and the microspheres can be loaded with MHC-2 and MHC-1 binding peptides (i.e., T epitope antigens) to produce a T and B cell mixed vaccine with antibody immunity and cellular immunity effects.

[0213] Example 9 Viral Therapeutic Vaccine

[0214] Many viral vaccines are on the market, the most successful of which are against smallpox, polio, and rabies. Other vaccines currently under development for the treatment of hepatitis, HIV, HPV, and the novel coronavirus are still in the clinical development phase and have not yet passed Phase III clinical trials before being marketed.

[0215] Similar to the case of tumor vaccines, the therapeutic efficacy of viruses is key. Vaccines prepared by traditional methods are not suitable for therapeutic use.

[0216] Taking COVID-19 as an example, the novel immune carrier microspheres of the present invention are applied. S protein antigen epitopes are coupled to the microsphere surface, and viral T cell epitopes are loaded inside the microspheres. Since the S protein is prone to mutation, multiple antigen peptides can be loaded, potentially increasing effective antibody titers by several orders of magnitude. Furthermore, the T epitopes are relatively stable and can be loaded in larger quantities, activating CD8 cells to kill infected cells and control infection.

[0217] COVID-19 receptor binding region (residues 319-541): contains the ACE2 receptor binding motif (RBM, residues 437-508), which is the main target of neutralizing antibodies.

[0218] Typical epitope sequences include residues K417, N439, G446, Y453, L455, F456, E484, and F490, which are bound by neutralizing antibodies (e.g., Regeneron antibody combination, LY-CoV555, etc.). E484 and N501 are frequently mutated in variant strains (e.g., Beta, Gamma, Omicron), potentially escaping antibody recognition.

[0219] The N-terminal domain (residues 14-26, 140-158): such as residues W152 and R246, is the target of certain neutralizing antibodies (such as 4A8 and COVA1-16).

[0220] Linear epitopes: such as residues 19-26 (NITNLCPF), residues 141-156 (GVGYQPYR), etc.

[0221] S2 subunit conserved region

[0222] Fusion peptide (FP) (residues 816-833): like residues S2P6 epitope (residues 1148-1159), conserved among multiple coronaviruses.

[0223] T cell epitopes are short peptides presented by MHC molecules and are divided into CD8+ (MHC-I) and CD4+ (MHC-II) epitopes.

[0224] The following are examples of validated T epitopes:

[0225] 1. CD8+ T cell epitopes (MHC-I restricted)

[0226] HLA-A*02:01 restricted epitopes: 269-27 (YLYALVYFL), 1291-1299 (NLDSKVGGN)

[0227] HLA-B*07:02 restricted epitope: 790-798 (SPRWYFYYL)

[0228] HLA-A*24:02 restricted epitope: 436-444 (NYNYLYRLF)

[0229] 2. CD4+ T cell epitopes (MHC-II restricted)

[0230] HLA-DRB1*01:01 restricted epitope: 685-694 (QIYKTPPIK)

[0231] HLA-DRB1*15:01 restricted epitope: 332-347 (IKLLEQFGNNKTFR)

[0232] Broad-spectrum epitope: S816-830 (GVTQNVLYENQKLI)

[0233] Effects of variants on epitopes:

[0234] Omicron (BA.1 / BQ.1 / XBB, etc.): A large number of mutations in the RBD region (such as K417N, E484A, N501Y) may escape B cell immunity, but T cell epitopes (such as the conserved region of the S2 subunit) are usually more conserved.

[0235] Cross-reactive T epitopes: such as the S2 subunit epitope of the S protein (such as 1167-1175 restricted by HLA-A*02:01), are highly conserved between SARS-CoV-1 and SARS-CoV-2.

[0236] The above is a detailed description of the embodiments to facilitate proper understanding and application of the present invention by those skilled in the art. Any improvements or modifications to the technical solutions derived by those skilled in the art based on the present invention, without inventive effort, solely through analysis, analogy, or limited enumeration, should be within the scope of protection defined by the claims.

Claims

1. A core-shell structure immune carrier microsphere constructed with personalized MHC-II binding polypeptide, characterized in that: The microspheres include a core and an outer shell; the core contains microspheres constructed from several individually designed MHC-II binding polypeptides, the MHC-II binding polypeptide sequences being obtained through computer prediction screening based on the HLA genotyping results of the vaccine recipient, and each HLA allele corresponds to at least one high-affinity MHC-II binding polypeptide; the outer shell is a thiol-containing dextran or other polymer coating layer, the thiol groups of which serve as coupling groups for connecting antigens to construct vaccines.

2. The core-shell structure immune carrier microspheres according to claim 1, characterized in that: The immune carrier microspheres contain 3-12 computer-screened high-affinity MHC-II binding polypeptides and at least one universal MHC-II binding polypeptide for assisting B cells in producing antibodies, wherein the universal MHC-II binding polypeptide is selected from at least one of the following: YFAVYLQETPY, AKFVAAWTLKAAA, QYIKANSKFIGITEL, FNNFTVSFWLRVPKVSASHLE, AWLEAQEEEEVGF, QYIKANSKFIGITELKK, EPRAPWIEQEGPEYWDQE, ADVEVYRAVTPLGPPD, DTLRSYYADWYQQKPG, FVNQHLAGSHLVEAL, LNEDLRSWTAADTAA.

3. The core-shell structure immune carrier microspheres according to claim 1, characterized in that: The MHC-II binding polypeptides are obtained by affinity scoring screening based on HLA detection typing using IEDB ProPred, MHCPred, SVRMHC, ARB, SMM-align, NetMHCIIpan, IEDB and NetMHCPan prediction software.

4. The core-shell immune carrier microspheres according to claim 1, characterized in that: The particle size of the microspheres is 0.1-10 microns. The microspheres are one of MHC-II binding polypeptide self-assembled microspheres, MHC-II binding polypeptide modified PLGA microspheres or albumin microspheres containing MHC-II binding polypeptides. The particle size of the MHC-II binding polypeptide self-assembled microspheres is 2000-5000 nm, the particle size of the MHC-II binding polypeptide coupled PLGA-PEG microspheres is 500-1000 nm, and the particle size of the HSA microspheres containing MHC-II binding polypeptides is 1-10 μm.

5. A method for preparing the immune carrier microspheres according to claim 1, characterized in that: The following steps are involved: S1: Individualized MHC-II binding peptide design: Based on the individual's HLA gene test results, computer software is used to screen high-affinity MHC-II binding peptide sequences; S2: Preparation of microsphere cores: The screened peptide sequences are synthesized into microspheres, and protein microspheres containing MHC-binding peptides are prepared by self-assembly, peptide-modified PLGA microspheres, or emulsification methods; S3: Shell coating: The core of the microsphere is coated with oxidized dextran with thiol or other polymer materials, which react with the core microspheres to form core-shell structure immune carrier microspheres.

6. A vaccine prepared based on the immune carrier microspheres described in any one of 1-4, characterized in that: The personalized MHC-II Binding peptide As immune carrier molecules Bag Enclosed in microspheres, the vaccine's antigen molecules are coupled to the surface of the microspheres to form a B cell microsphere vaccine.

7. The vaccine prepared by the immune carrier microspheres according to claim 6, characterized in that: The microspheres replace KLH, bacteriophage, and viral immune carrier molecules and are used to prepare new vaccines using endogenous proteins or polypeptides.

8. The vaccine prepared by the immune carrier microspheres according to claim 6, characterized in that: The microspheres are loaded with MHC-II and T cell epitope polypeptides, and are used to prepare T and B mixed vaccines with antibody immunity and cellular immunity effects.

9. The vaccine prepared by the immune carrier microspheres according to claim 6, characterized in that: The surface of the microspheres is coupled with a variety of different antigens to construct new vaccines.

10. Use of vaccines prepared from the immune carrier microspheres as described in claims 6-9 in the preparation of drugs for tumors, cardiovascular diseases, hyperglycemia, allergic diseases, autoimmune diseases, Alzheimer's disease, viral infections, drug addiction treatment, weight loss and other chronic diseases that can be treated with antibodies.

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