A method for multi-antigen epitope merging recombination based on computer molecular simulation technology

Abstract

The application discloses a method for preparing multi-antigen epitope peptides based on computer molecular simulation technology. In the method, the key amino acid combination of each monoclonal antibody is analyzed first, and then the key amino acid of each monoclonal antibody is combined and recombined into thousands of multi-antigen epitope peptides with different sequences under the guidance of computer molecular simulation technology. Meanwhile, the variable region amino acid sequence of each monoclonal antibody is analyzed, and a three-dimensional structure model of the variable region of each monoclonal antibody is constructed through computer molecular simulation technology. Finally, the model is used to simulate the binding of each recombined multi-antigen epitope peptide, and the multi-antigen epitope peptide which can be well combined with each monoclonal antibody is screened out for biological experiment identification. The multi-antigen epitope peptide which has good affinity and specificity to each monoclonal antibody is screened out through biological experiment, and is used for subsequent work such as vaccine preparation, diagnostic product production or drug research and development.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of immunology, and particularly relates to a method for recombining immunodominant groups of multiple antigen epitopes under the guidance of computer molecular simulation technology, and screening multiple antigen epitope peptides by taking antibody variable regions or viral receptor proteins as templates. BACKGROUND

[0002] Immunity is initiated by antigen stimulation of the body, and the antigen can stimulate the immune system of the body to produce an immune response, and can bind with the immune response product (antibody or sensitized lymphocyte) [1-3]. It is currently believed that when an antigen molecule exerts an immune stimulating effect, it does not exert the effect as a complete molecule, but exerts the stimulating effect on immune cells or the binding effect with antibodies as some local structure of the antigen molecule. Such a local structure of the antigen molecule is called an antigenic determinant, or an antigenic epitope [4, 5]. It is currently generally believed that a polypeptide epitope contains 5-6 amino acid residues; a polysaccharide epitope contains 5-7 monosaccharides; and a nucleic acid antigen epitope contains 6-8 nucleotides [6, 7]. It is found in polypeptide antigens that one group plays a greater role than other amino acid residues in binding with antibodies, and such a group is called a key amino acid. People have conducted a large number of studies on the key amino acids of various different polypeptide epitopes in the same way, but in their reports, the key amino acid of the antigen epitope is not one, but a combination [8]. With the development of various sequencing technologies, protein recombination technologies and crystal diffraction technologies, a large number of databases, models and algorithms related to antigens have been established [7, 9]. Although we have learned a lot about the interaction between antigens and antibodies, what is the nature of the antigen epitope? There is still no conclusion. What kind of antigen epitope can induce the human body to produce an immune response against self antigens? How can a "universal" vaccine be made for viruses that often mutate, such as the new coronavirus, and other problems all depend on our further understanding of the composition of the antigen epitope.

[0003] With the development of sequencing technology, computational technology and protein structure analysis technology, more and more biological databases have been constructed. When considering the analysis of a specific antigen epitope, one can either use polypeptide superposition expression or first use computer to predict and then use biological experiments to further confirm the function of these epitopes. For example, Guevarra used computer analysis tools to predict the specific immunogenic epitope of dengue virus type 2 NS1 antigen, and synthesized the mimetic peptide to immunize rabbits. It was found that the rabbit produced antibodies that could specifically bind to the predicted mimetic peptide synthesized according to the unique DENV2 NS1 immunogenic epitope

[13] . Javadi used computer models and immunoinformatics tools to design effective microbial antigen multi-epitopes, especially to collect various antigen immunogenic epitopes, thus making high immunogenicity multi-epitope recombinant antigens; these research results are of great significance for disease prevention and diagnostic reagent development

[14] . At present, people use different research methods from different angles to meet different needs, and have established various methods or online tools for antigen epitope modification and design, such as ABCpred (Artificianeural network based B-cell epitope prediction server), LBtope (Linear B-cell Epitope Prediction server), IEDB (The Immune Epitope Database), BCpred (Prediction of Continuous B-Cell Epitopes), SVMTriP (Support Vector Machine to integrate Tri-Peptide) and PEPOP, etc. [15-20]. With the help of these tools, in recent years, scientists have made a lot of meaningful research results in the study of 2 type dengue virus, Zika virus, chikungunya virus, hantavirus and toxoplasma-related epitopes by combining online tool prediction with animal model verification [13, 14, 21-23]. However, due to the lack of a unified understanding of the composition and characteristics of antigen epitopes, so far, there is no unified software programming idea and no universally recognized polypeptide antigen epitope prediction model. Different software can only focus on analyzing antigen epitopes from their own correct perspective, which not only indicates the complexity of antigen epitopes, but also shows the urgency and necessity of in-depth study of the nature of antigen epitopes.Moreover, the research results of the above literatures are all based on the amino acid sequence of the original protein itself, and there is no research report on the synthesis of non-homologous multi-antigen epitope polypeptides with the original protein by computer simulation technology to carry out antigen epitope prediction, merger recombination or application.

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[0027] In order to solve the above problems, the application provides a screening of immune dominant base combination based on computer molecular simulation and application thereof in preparation of recombinant multi-antigen epitope peptide.

[0028] The technical scheme adopted by the application is as follows.

[0029] The application provides a multi-antigen epitope combination and recombination method based on computer molecular simulation technology and antigen epitope key amino acid (immune dominant base) combination theory.

[0030] S1, the number of single-antibody combined epitopes to be combined and recombined is determined, and the key amino acid combination of the epitope combined by each single-antibody is analyzed.

[0031] S2, under the guidance of computer molecular simulation technology, all the key amino acids of the planned combined antigen epitopes are combined and recombined to recombine a multi-antigen epitope peptide containing multiple single-antibody combined epitopes.

[0032] S3, mRNA of the single-antibody hybridoma cell strain is retrieved, cDNA is synthesized by reverse transcription, the light-chain variable region gene and the heavy-chain variable region gene of each single-antibody are amplified respectively, and the single-antibody variable region amino acid sequence is obtained; the three-dimensional structure model of the single-antibody variable region is constructed by computer molecular simulation technology.

[0033] S4, each single-antibody is combined with each recombined multi-antigen epitope peptide by polypeptide-protein docking through computer molecular simulation technology, and the combination is simulated; the binding energy between the recombined multi-antigen epitope peptide and each single-antibody obtained by molecular simulation combination is ranked, and the recombined multi-antigen epitope sequence which is possibly well combined with each single-antibody is screened and recommended.

[0034] S5, the screened and recommended multi-antigen epitope peptide is subjected to polypeptide synthesis and biological identification, so as to determine whether the design requirement is met.

[0035] In the S1, the key amino acid combination of each monoclonal antibody binding epitope is analyzed by replacing the amino acid in the polypeptide with alanine one by one, then synthesizing the alanine replacement peptide, calculating the inhibition index by ELISA blocking experiment, and determining whether the replaced amino acid is a key amino acid according to the inhibition index.

[0036] The influence of the replaced amino acid in the polypeptide on the binding of the polypeptide and the monoclonal antibody is detected by the ELISA blocking method; if the replaced amino acid has a great influence on the binding of the polypeptide and the antibody, it is indicated that the replaced amino acid plays a greater role in the binding of the polypeptide and the antibody, and is a key amino acid; otherwise, it is a non-key amino acid.

[0037] In the S2, a plurality of multi-epitope peptides are randomly recombined from the key amino acids of each antibody binding by computer molecular simulation technology (the number of recombined multi-epitope peptides is different due to the different number of combined monoclonal antibody binding epitopes), and ranking is performed based on the physicochemical properties and geometric fragment similarity of the polypeptide; as shown in an embodiment of the present patent, 4608 polypeptides are randomly recombined from the key amino acids of each antibody binding, and finally 61 artificial recombinant multi-epitope peptides with potential binding activity sites of 6 monoclonal antibodies are obtained by ranking.

[0038] In the S3, the crystal protein with the best similarity to the amino acid sequence of each monoclonal antibody is selected from the RCSB PDB protein crystal database as a template for homology modeling, and the variable region three-dimensional structure model of each monoclonal antibody is constructed by using the homology modeling software Modeller.

[0039] In the S4, the UCSF Chimera software is used to construct the three-dimensional structure of the target antigen peptide of each monoclonal antibody, and the Rosetta software is used to perform polypeptide-protein docking of each monoclonal antibody with the multi-epitope peptides ranked at the top in the S2 step, and the polypeptides with good binding activity to each monoclonal antibody and ranked at the top are screened for polypeptide synthesis and biological verification; for example, in an embodiment of the present patent, 6 recombinant multi-epitope peptides with an affinity greater than -8.0 Kcal / mol to each monoclonal antibody are obtained after simulating the binding of the variable regions of 6 antibodies to 61 multi-epitope peptides.

[0040] In the S5, the recommended recombinant multi-epitope peptide also needs to be evaluated in the laboratory for its binding activity to each monoclonal antibody, the blocking effect on the binding of the antibody and the target antigen, the cross-reactivity with the antibody, and other biological indicators required for the design purpose, and the multi-epitope peptide with good evaluation results and meeting the design purpose is selected for further research or application.

[0041] Further, in the S2, the step of splitting, combining, and recombining the amino acids of the key amino acid combination of the single antibody binding by computer molecular simulation technology includes:

[0042] Step A: using Cd-hit software to perform sequence similarity matching on the key amino acids of the single antibody binding antigen epitope peptide;

[0043] Step B: according to the geometric shape and physicochemical properties of the amino acids, performing global physicochemical property matching on the single antibody binding antigen epitope peptide;

[0044] Step C: geometric configuration similarity matching;

[0045] Step D: similarity amino acid matching;

[0046] Step E: scoring and ranking the thousands of randomly recombined multi-antigen epitope peptides based on physicochemical properties and geometric fragment similarity, and finally obtaining dozens or hundreds of multi-antigen epitope peptides with potential binding activity sites of multiple single antibodies.

[0047] The "key amino acid" in the application refers to an amino acid on an antigen that plays a key role when binding with an antibody; the "antigen epitope" refers to a part of the antigen that binds with the antibody as a whole, a local structure on the antigen; and the "key amino acid combination" refers to the part of the antigen that binds with the antibody as several amino acids that jointly play a role, or a combination of groups thereon.

[0048] The obtained recombinant multi-antigen epitope peptide with the function of inducing anti-influenza HA immune response has the sequence ILLWVLSHL.

[0049] The screening method is applied to finding antigen epitopes with similar antibody functions induced by different structures or sequences.

[0050] The screening method is applied to the preparation of a polypeptide vaccine, a multivalent vaccine, or a polypeptide drug.

[0051] The multi-antigen epitope combination and recombination method can be used to recombine the neutralizing epitopes of multiple viruses to prepare a multivalent vaccine when the screened antibody is replaced by a receptor protein of a virus; the neutralizing epitopes of viruses such as the new coronavirus and HIV virus, which have weak immunogenicity, are recombined, and according to the antibody cross-reaction theory, a polypeptide vaccine that can easily stimulate the corresponding neutralizing antibodies of the virus can be developed; similarly, a polypeptide drug with weak immunogenicity can also be screened and developed by the method.

[0052] The application relates to a method for obtaining an artificial recombinant polyepitope peptide, which takes polypeptides combined with monoclonal antibodies of target virus proteins with different reaction characteristics as research objects, splits, combines and recombines key amino acid combinations of the monoclonal antibodies on the target virus antigen polypeptides under the guidance of computer molecular simulation technology, takes the variable region of the monoclonal antibodies as a template to screen the recombinant polyepitope peptide, and obtains the artificial recombinant polyepitope peptide.

[0053] The recombinant polyepitope peptide is screened by splitting, combining and recombining amino acids of the key amino acid combinations of the monoclonal antibodies under the guidance of computer molecular simulation technology; and the recombined polyepitope peptide is simulated to be combined with the variable region modeling structure of the monoclonal antibodies, so that the recombinant polyepitope peptide which can be well combined with the monoclonal antibodies is screened out.

[0054] Taking the influenza virus as an example, the method for combining and recombining the polyepitope based on the computer molecular simulation technology is described, and the method comprises the following steps:

[0055] 1) two polypeptides are intercepted from the influenza virus HA protein, polypeptide 1: 191-LVLWGIHHP-199; polypeptide 2: 93-WSYIVE-98, and key amino acids are determined.

[0056] 2) under the guidance of computer molecular simulation technology, all the key amino acids of the planned combined antigen epitopes are combined and recombined to recombine the polyepitope peptide containing multiple monoclonal antibody binding epitopes.

[0057] 3) mRNA of the hybridoma cells combined with the target virus antigen is reversely transcribed to synthesize cDNA, which is used as a template for subsequent gene cloning; the monoclonal antibody light chain variable region gene and the monoclonal antibody heavy chain variable region gene are amplified respectively to obtain amino acid sequences; a computer molecular simulation technology is used to construct a three-dimensional structure model of the monoclonal antibody variable region; the best crystal protein with the highest similarity to the amino acid sequence of each monoclonal antibody is selected from RCSB PDB protein crystal database as a template for homology modeling, and a homology modeling software Modeller is used to construct a three-dimensional structure model of each monoclonal antibody variable region.

[0058] 4) the computer molecular simulation technology is used to analyze the protein-protein docking of the monoclonal antibodies and the polypeptide fragments; the recombinant polyepitope peptide screened out is used for protein-polypeptide docking with the monoclonal antibodies by using Rosetta software, and the binding energy and the ranking between the polypeptide and each monoclonal antibody are obtained, and the polypeptide with good binding activity and ranking at the front is selected; the Rosetta software is used for protein-protein docking of the six monoclonal antibodies and the polypeptides WSYIVE and LVLWGIHHP, and the protein-protein docking adopts a global docking method, the polypeptide is evenly distributed on the spherical polypeptide binding pocket with the receptor protein as the center of the spherical system.

[0059] 5) Recombinant polyepitope peptides that bind well to the monoclonal antibodies are screened based on surface accessibility, hydrophilicity, and affinity.

[0060] In Step 2), the steps of splitting, combining, and recombining the amino acids of the key amino acid combination bound by the monoclonal antibody are performed by computer molecular simulation technology, including:

[0061] Step A: Sequence similarity matching of key amino acids of dipeptides LVLWGIHHP and WSYIVE is performed using Cd-hit software;

[0062] Step B: Global physicochemical property matching of polypeptides LVLWGIHHP and WSYIVE is performed according to the geometric shape and physicochemical properties of the amino acids;

[0063] Step C: Geometric configuration similarity matching;

[0064] Step D: Similarity amino acid matching;

[0065] Step E: Scoring and ranking of 4608 polypeptides based on physicochemical properties and geometric fragment similarity, and finally obtaining 61 artificially combined polypeptides with the potential to simultaneously bind to the active sites of 6 monoclonal antibodies.

[0066] After the above steps, the recombinant polyepitope peptides that induce anti-influenza HA immune response are screened, and the sequences are ILLWVLSHL, LLIWVLSHL, ILIWVLSHI, ILIWVLSHV, ILIWVISHL, and ILIWVLSHL.

[0067] It is generally accepted that the epitope of an antigen is the smallest functional unit of the antigen that can elicit immune response. The binding of an epitope to an antibody is a one-to-one relationship, and the epitope determines the specificity of the antigen. According to the classical immunology, the B cell recognizes the linear or conformational epitope of the native antigen through its B cell receptor. The composition of the epitope is specific, and the linear epitope is usually determined by the amino acid sequence of the antigen itself. This is the basis of the current vaccine design. Our research group found that several monoclonal antibodies against the HA protein of the influenza virus were localized on the same peptide segment, but the amino acid sequences of the variable regions of these monoclonal antibodies were not the same, indicating that these monoclonal antibodies were not secreted by a single hybridoma cell. Moreover, the reaction characteristics of the three monoclonal antibodies that bound to the same peptide segment were not the same, suggesting that the three monoclonal antibodies may not bind to the same epitope on the same short peptide. That is, although the peptide segment is short, it may also form more than one epitope. Through alanine scanning amino acid substitution method to analyze the binding epitope of the antibody, it was found that there were 1-2 amino acid differences between the key amino acid combinations of each monoclonal antibody, suggesting that the reason for the different reaction characteristics of the three monoclonal antibodies that bind to the same short peptide may be the difference in the key amino acid combinations they bind to. That is, these antibodies may be stimulated by different key amino acid combinations. This result indicates that the basic functional unit of a polypeptide antigen, the epitope, may be a combination of key amino acids.

[0068] To verify whether a small molecule short peptide can form multiple epitopes, we prepared monoclonal antibodies against two peptide segments LVLWGIHHP and WSYIVE from the HA protein of the influenza virus. The results showed that some of the monoclonal antibodies stimulated by these peptides could react with the HA antigen, and some could not. Moreover, the reaction characteristics of the monoclonal antibodies that bound to the HA antigen were not the same as those that did not bind to the HA antigen, suggesting that a small molecule of 6 peptides may also form multiple different epitopes. These results suggest that the epitope may not be a fixed structure, but a combination of multiple amino acid groups (these groups that play an immune stimulating role are called immunodominant groups). If so, the groups in the key amino acid combination that make up the epitope can be replaced by groups with the same or similar properties. That is, the key amino acid combination that makes up the epitope can be split and recombined.

[0069] The fact that at least 3 or more single antibody binding epitopes are concentrated on such a short peptide segment indicates the complexity of the antigen epitope composition. The above research results show that the binding of small molecule polypeptides to antibodies is actually the interaction between amino acids (groups) on the polypeptide chain and the groups on the amino acid residues of the antibody variable region. Whether they can bind depends on their surface accessibility and affinity to each other; if these conditions are met, the immunodominant groups in the polypeptide can bind to different antibodies in different combinations. That is, the groups of amino acids in the polypeptide can all act as immunodominant groups when immunized. These immunodominant groups can act as different antigen epitopes in different combinations to stimulate immunity. Which or which combination of these combinations stimulates immunity depends on the selection of immune cells.

[0070] On the basis of the previous research, analysis and understanding of polypeptide antigen epitopes, the research group proposed the view that polypeptide antigen epitopes are combinations of immunodominant groups, each immunodominant group in the combination can be replaced by an immunodominant group of the same nature, and multiple antigen epitopes can be combined and recombined into a multi-antigen epitope peptide. To verify the correctness of this view, the research used computer molecular simulation technology to split and recombine the key amino acid combinations bound by the 6 monoclonal antibodies into a multi-antigen epitope peptide. The variable regions of the 6 monoclonal antibodies were subjected to protein structure homology modeling, and the binding activity of the recombined multi-antigen epitope peptide to the 6 monoclonal antibodies was analyzed under the guidance of computer molecular simulation technology; it was proved that although the sequences of some recombined multi-antigen epitope peptides were different from those of the polypeptides LVLWGIHHP and WSYIVE, the recombined multi-antigen epitope peptides did carry antigen epitopes that could bind to the 6 monoclonal antibodies. After mice were routinely immunized with the BSA-labeled recombined multi-antigen epitope peptide ILLWVLSHL, strong antibodies against polypeptides LVLWGIHHP and WSYIVE and H1N1 influenza virus antigen were induced. This proves that the antigen epitopes on the two polypeptides and the H1N1 antigen HA protein were all transferred to the recombined multi-antigen epitope peptide using the 6 monoclonal antibodies as a template, that is, there are similar antigen epitopes on the recombined multi-antigen epitope peptide to those on the two polypeptides and the H1N1 influenza virus antigen. The result that the antibody titer of the recombined multi-antigen epitope peptide immune serum against the H1N1 influenza virus antigen is higher than that of the antibodies against polypeptides LVLWGIHHP and WSYIVE seems to suggest that the recombined multi-antigen epitope peptide ILLWVLSHL carries more identical antigen epitopes to those on the H1N1 influenza virus antigen than the two polypeptides LVLWGIHHP and WSYIVE each carrying identical antigen epitopes. From this, we established a method of recombining multi-antigen epitopes under the guidance of computer molecular simulation technology and screening multi-antigen epitopes using antibody variable regions as a template; and it was proved that the recombined peptide carrying multi-antigen epitopes can also well display its immunogenicity when immunized.

[0071] The present study proposes the concept of polypeptide epitope as a combination of immunodominant bases, which believes that the antigen epitope is a combination of immunodominant bases, and the immunodominant bases in the combination can come from adjacent amino acids or from non-adjacent amino acids that are far apart in the amino acid sequence of the protein; these immunodominant bases can be combined in different combinations with different immune cells to exert different immune stimulation effects; as for which combination of immunodominant bases exerts immune stimulation, it completely depends on the selection of antigen receptors on immune cells.

[0072] On the other hand, we synthesized polypeptides that can induce the production of anti-influenza virus HA antibodies through computer molecular simulation technology, which is different from the amino acid sequence of the influenza virus antigen. The establishment and practice of this new concept provide new theoretical and application research ideas for the research and development of polypeptide vaccines, multivalent vaccines, and polypeptide drugs. The mutation of amino acids in the key parts of the new coronavirus that began to spread at the end of 2019 affects the immune effect of the vaccine, and our single polypeptide multi-epitope loading concept will be beneficial to the design and application of new coronal vaccines. In addition, the method in this paper can also be used for other researches, such as in the research of autoimmune diseases, we will abandon the previous attempt to find the corresponding relationship between the existing antigen epitope library and human body's own antigen epitope, and use the concept of immunodominant base combination to find antigen epitopes that are different in structure or sequence but induce similar antibody functions. In addition, our method can also be used in the discovery of tumor-related (specific) antigens and subsequent vaccine development, the identification of new polypeptide-related diagnostic epitopes, and the research and development of polypeptide drugs.

[0073] The screening method of the present application is used in the preparation of polypeptide vaccines, multivalent vaccines or polypeptide drugs. Specifically, for example, due to the variability of the new coronavirus, the key amino acid combinations used by the five subtypes of Alpha, Beta, Gamma, Delta and Omicron viruses to bind to ACE2 are split and recombined into many multi-antigen epitope peptides under the guidance of computer molecular simulation technology, and then the ACE2 receptor protein crystal model combined with each multi-antigen epitope peptide is simulated to screen various polypeptides that can bind to ACE2 receptor. Mark the carrier, immunize the mouse, detect the immune serum by biological neutralization test, determine the serum with high titer that can block the binding of five main subtypes of viruses to ACE2, and use the multi-antigen epitope peptide for preparing the serum as a new coronavirus vaccine, so as to develop a multivalent vaccine against five subtypes of new coronaviruses with strong antigenicity.

[0074] For vaccines like COVID-19 and HIV, which have weak immunogenicity and are unlikely to stimulate the production of neutralizing antibodies or produce neutralizing antibodies with short durations of action, recombinant modification of natural vaccines can be employed. For example, the 16 amino acids on the spike protein of the SARS-CoV-2 virus, which bind to the ACE2 receptor protein, can be recombined using computer molecular simulation technology. Then, the recombinant peptides can be simulated to bind to the ACE2 receptor protein, allowing for the selection of peptides that bind well to the receptor protein. These peptides can then be labeled with a vector, routinely immunized in animals, and the titer of neutralizing antibodies against SARS-CoV-2 in the immune serum can be measured. Recombinant peptides that stimulate high-titer neutralizing antibodies can be selected for vaccine preparation, thereby achieving the goal of preventing SARS-CoV-2 infection and stopping its spread.

[0075] Beneficial effects:

[0076] This invention utilizes computer simulation technology to synthesize multi-antigenic epitope peptides that are not homologous to the original protein for epitope prediction, merging, and recombination. It proposes for the first time that an epitope is a combination of immunodominant groups, each of which can be replaced by an immunodominant group of the same nature, and multiple epitopes can be merged and recombine to form a single multi-antigenic epitope peptide. This invention establishes a method for merging and recombinating multi-antigenic epitopes under the guidance of computer molecular simulation technology, and for screening multi-antigenic epitopes using antibody variable regions as templates. Furthermore, it demonstrates that recombinant peptides carrying multi-antigenic epitopes can effectively exhibit immunogenicity during immunization. The establishment and practice of this new concept provide new theoretical and applied research ideas for the development of peptide vaccines, multivalent vaccines, and peptide drugs. Attached Figure Description

[0077] Figure 1 Computer simulation flowchart of recombinant peptides;

[0078] Figure 2 Schematic diagram of the binding pattern between mAbs and various peptide molecules and the energy of intermolecular interactions (kcal / mol); mAbs: light cyan; peptides: blue; amino acids within 0.4 nm between the mAbs and peptides are the key amino acids for interaction;

[0079] Figure 3 The blocking effect of recombinant multi-antigen epitope peptides on the binding of monoclonal antibodies to influenza virus antigens; Figure caption: Each line in the figure shows the blocking effect of each recombinant multi-antigen epitope peptide on the binding of each monoclonal antibody to influenza virus antigens. The results in the figure show that XH-001 and XH-006 have good blocking effects on each monoclonal antibody. Detailed Implementation

[0080] Example 1

[0081] 1. Materials:

[0082] 1) Influenza virus strains: Influenza A H1N1 virus split vaccine (A / reassortant / NYMC X 179A (California / 07 / 2009 x NYMC X 157) (H1N1)) (National Drug Standard Number S20090015) was purchased from Walvax Biotech Co., Ltd.; Seasonal H1N1 (A / Brisbane / 59 / 2007 (H1N1)), 501 strain, Pr8 strain antigens were prepared by the laboratory; H3N2 influenza vaccine (A / Victoria / 210 / 2009 (H3N2)) was a gift from Dalian Yali Feng Biotechnology Co., Ltd.; H5N1 (A / Goose / Guangdong / 1 / 96 (H5N1)) veterinary drug (2009); H7N2 antigen was a gift from the China Animal Disease Prevention and Control Center; H7N2 and H7N9 antigens were a gift from the China Disease Prevention and Control Center.

[0083] 2) Antibodies: Six monoclonal antibodies used in this paper, including A1-10, H1-74, H1-84, H1-5, H1-16, H1-81, etc., were anti-H1N1 influenza virus HA protein monoclonal antibodies prepared by the laboratory according to the literature

Guo C, Xie X, Li H, Zhao P, Zhao X, Sun J, Wang H, Liu Y, Li Y, Hu Q et al: Prediction of common epitopes on hemagglutinin of the influenza A virus (H1 subtype). Exp Mol Pathol 2015, 98(1): 79-84

[0084] 3) Polypeptides: All polypeptides used in the experiment were synthesized by Shanghai Qiangyao Biotechnology Co., Ltd., and the products were detected by HPLC and MS with a purity of >85%. Two polypeptides were cut from the influenza virus HA protein: polypeptide 1, 191-LVLWGIHHP-199; polypeptide 2, 93-WSYIVE-98. Using these two polypeptides as templates, alanine was used to replace the amino acids in the polypeptides one by one to synthesize alanine replacement peptides.

[0085] 4) Other reagents: Fetal bovine serum (cat. no. 16000-044) for cell culture was purchased from Hangzhou Sijiqing Biological Engineering Material Co., Ltd.; total RNA extraction kit (cat. no. DP433), cDNA first strand synthesis kit (cat. no. KR104) and DH5a competent cells (cat. no. CB101) were purchased from Tiangen Biotech Co., Ltd. 6cfu / μg, PCR polymerase (cat. no. C10966-018), pMD19-T vector (cat. no. 6013) and DNA Marker (cat. no. D526A) were purchased from Takara Bio Co., Ltd. (Dalian, China). Primer synthesis and sequencing were completed by Beijing Huada Gene Technology Co., Ltd. Mouse myeloma cells (Sp2 / 0) were purchased from ATCC. BALB / c mice (8 weeks old, female) were purchased from the Experimental Animal Center of Air Force Medical University. Polyethylene glycol (PEG) (cat. no. 57354-U) was purchased from Sigma. mAb Subtype Identification Kit SBAClonotyping TM System / HRP (cat. no. 5300-05) was a product of Southern Biotech, and HAT (cat. no. 21060017) and 1640 cell culture medium containing bovine serum (cat. no. C11995500BT) were purchased from Gibco.

[0086] 2. Determination of key amino acids of antigen epitopes

[0087] 1) Antigen-antibody reaction and indirect ELISA experiment

[0088] Various antigens, including 2009 H1N1 influenza virus split vaccine, H1N1 01 strain and Pr8 strain, seasonal influenza virus H3N2 vaccine, avian influenza H5N1, H7N2 virus, H7N9 virus, and cross-linked polypeptide, were coated overnight at 4°C. After washing, the culture supernatant of hybridoma cells was added as the primary antibody, and allantoic fluid and SP2 / 0 supernatant were used as negative controls. The reaction was carried out at 37°C for 1 h. After washing, HRP-labeled goat anti-mouse secondary antibody (1:2500 dilution) was added, and the reaction was carried out at 37°C for 1 h. After washing, TMB color developing solution was added for color development, and the A450nm OD value was measured on an ELISA reader. A positive / negative (P / N) ratio >2.5 was considered as a positive result.

[0089] 2) Alanine scanning peptide design and preparation

[0090] The amino acids of polypeptides LVLWGIHHP and WSYIVE were replaced with alanine one by one, and then alanine substitution scanning peptides were synthesized (alanine substitution scanning peptides of the two antigen peptides were synthesized by Shanghai Qiangyao Biological Company), a total of 17 (see Table 1).

[0091] Table 1. Alanine scanning substitution peptide sequences of HA protein

[0092]

[0093] 3) Determination of key amino acids and ELISA blocking experiments

[0094] H1N1 influenza virus HA antigen was coated on 96-well ELISA plates at a concentration of 2-5 ug / ml, 4°C overnight, washed, and blocked for later use. The appropriate dilution of monoclonal antibodies was first reacted with various polypeptides, respectively, at 37°C for 1 h, and then added to the pre-coated HA ELISA plates, followed by the conventional indirect ELISA operation steps, and finally TMB color developing solution was added for color development, and the A450nm OD value was measured on the ELISA reader. According to the OD450nm value of each well, the inhibition index was calculated according to the formula: Inhibition index IR = (OD value of the control group without polypeptide - OD value of the experimental group with polypeptide) / OD value of the control group without polypeptide. Inhibition index ≤ 40% indicates that the antigen peptide has weak binding activity with the antibody, and the replaced amino acid in the antigen peptide is a key amino acid; Inhibition index between 40% and 80% indicates that the antigen peptide has certain binding activity with the antibody, indicating that the replaced amino acid has a certain influence on the binding of the antibody and the antigen; Inhibition index ≥ 80% indicates that the antigen peptide has strong binding activity with the antibody, and the replaced amino acid does not affect the binding of the antigen peptide and the antibody, and the replaced amino acid is a non-key amino acid.

[0095] 3. Analysis of the structure of monoclonal antibodies and their interaction with polypeptides

[0096] 1) Sequencing of the variable region genes of monoclonal antibodies

[0097] Method according to the literature

[10] (Guo C, Xie X, Li H, Zhao P, Zhao X, Sun J, Wang H, Liu Y, Li Y, Hu Q et al: Prediction of common epitopes on hemagglutinin of the influenza A virus (H1 subtype). Exp Mol Pathol 2015, 98(1): 79-84), mRNA of hybridoma cells combined with influenza virus HA antigen was extracted, reverse transcribed to synthesize cDNA, which was used as a template for subsequent gene cloning. A total of 27 primers were synthesized by comparing and analyzing the variable region gene sequences of mouse monoclonal antibody light chain and heavy chain published by NCBI

[10] The single antibody light chain variable region gene was amplified by 8 primers, and the single antibody heavy chain variable region gene was amplified by 9 primers. The light chain gene was about 320 bp, and the heavy chain gene was about 350 bp. The PCR amplified product was subjected to PCR identification again. The positive band of light chain identification was about 180 bp, and the positive band of heavy chain identification was about 150 bp. The positive fragments obtained by PCR amplification were recovered and connected with pMD19-T vector by a conventional method. The connection product was transformed into E. coli DH5a competent cells, and the monoclonal bacteria were picked, the plasmid was extracted and identified by enzyme digestion, and after sequencing, the obtained nucleotide sequence was translated into amino acid sequence.

[0098] 2) Construction of the three-dimensional structure model of the variable region of the single antibody and its interaction with the polypeptide

[0099] The crystal protein with the best similarity to the amino acid sequence of each single antibody was selected from the RCSB PDB protein crystal database as a template for homology modeling, and the three-dimensional structure model of the variable region of each single antibody was constructed using the homology modeling software Modeller.

[0100] The UCSF Chimera software was used to construct the three-dimensional structure of the polypeptides WSYIVE and LVLWGIHHP. The Rosetta software was used to perform protein-protein docking of the 6 single antibodies with the polypeptides WSYIVE and LVLWGIHHP, respectively. The docking method was global docking, and the receptor protein was taken as the center of the spherical system, and the polypeptide was evenly distributed on the polypeptide binding pocket. The docking in Rosetta includes two steps: (1) forming a larger docking binding site around the amino acid residues of the active site of the receptor, and then scanning with different types of atoms as probes to calculate the grid energy; (2) searching for the conformation of the ligand in the Box range, and finally scoring according to the different conformations, directions, positions and energies of the ligand, and ranking the results. (3) 100 polypeptide systems were subjected to protein-polypeptide molecular docking, 100 docking phases were obtained, and energy ranking was performed. The lowest energy phase of WSYIVE and LVLWGIHHP binding to the 6 antibodies was selected as the best representative of each system for subsequent analysis.

[0101] 3) Preparation of carrier protein BSA and KLH coupled small molecule polypeptide and single antibody

[0102] The polypeptide carboxylic acid end was activated by EDC activator (1-(3-dimethylaminopropyl)-3-ethyl carbodiimide), and then coupled (cross-linking method provided by Shanghai Qiangyao Biological Company) with carrier protein bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH).

[0103] The polypeptides LVLWGIHHP and WSYIVE cross-linked at the carboxyl end were emulsified with Freund's complete adjuvant and subcutaneously immunized BALB / c mice at a dose of 20-25 μg per mouse. Three weeks later, the mice were boosted 2-3 times with the same dose of antigen and Freund's incomplete adjuvant. Three days before cell fusion, the mice were intraperitoneally boosted with antigen without adjuvant. Cell fusion and screening of positive hybridoma cells were performed according to the method described in the literature

[10] The polypeptides LVLWGIHHP and WSYIVE cross-linked at the carboxyl end were emulsified with Freund's complete adjuvant and subcutaneously immunized BALB / c mice at a dose of 20-25 μg per mouse. Three weeks later, the mice were boosted 2-3 times with the same dose of antigen and Freund's incomplete adjuvant. Three days before cell fusion, the mice were intraperitoneally boosted with antigen without adjuvant. Cell fusion and screening of positive hybridoma cells were performed according to the method described in the literature

[0104] 4. Recombination and screening and functional test of multi-epitope peptide

[0105] 1) Recombination of multi-epitope peptide

[0106] The amino acids of the 6 key amino acid combinations bound by the 6 anti-influenza virus monoclonal antibodies were split, combined and recombined by computer molecular simulation technology. The recombined multi-epitope peptides were simulated to bind to the variable region modeling structure of the 6 monoclonal antibodies, respectively. The surface accessibility, hydrophilicity and affinity were used as indicators to screen the artificial recombinant multi-epitope peptides that can bind well to the 6 monoclonal antibodies. The detailed process is as follows:

[0107] The key amino acids of the 6 monoclonal antibodies bound to the 2 polypeptides LVLWGIHHP and WSYIVE were mixed and recombined to form artificial recombinant multi-epitope peptides with combined antigen epitopes

[0108] Step A: The key amino acids of the dipeptides LVLWGIHHP and WSYIVE were matched for sequence similarity using Cd-hit software. The polypeptide amino acid sequences and active amino acid sites on each antibody are as follows:

[0109] (1) A1-10: LVLWGIHHP

[0110] (2) H1-84: LVLWGIHHP

[0111] (3) H1-74: LVLWGIHHP

[0112] (4) H1-16: WSYIVE

[0113] (5) H1-81: WSYIVE

[0114] Among them, the co-related critical amino acids (accounting for >60%) bound by monoclonal antibodies A1-10, H1-84, H1-74 on the polypeptide are: LVLWGIHHP; the co-related critical amino acids (accounting for >60%) bound by monoclonal antibodies H1-5, H1-16, H1-81 on the polypeptide are: WSYIVE.

[0115] Step B: According to the geometric shape and physicochemical properties of amino acids, the polypeptides LVLWGIHHP and WSYIVE are globally matched in physicochemical properties, obtaining a total of 559832 LVLWGIHHP amino acid combinations and 1728 WSYIVE random combinations. Rank the sequence similarity of 1728 WSYIVE amino acid fragments and 559832 LVLWGIHHP amino acids. It is found that due to the problem of SYE in WSYIVE, the sequence similarity between them cannot reach 50%, so similarity matching scoring cannot be performed.

[0116] Step C: Geometric configuration similarity matching. E in WSYIVE peptide segment is a negative charged amino acid, which is completely opposite to the electric negativity of H (positive charged) amino acid on LVLWGIHHP, so it is finally placed on both sides of the combined peptide segment, or directly deleted and screened out in sequence matching. In WSYIE, since SY has no relevance with any amino acid in LVLVWGIHHP, this amino acid needs to be analyzed in detail. It is extremely similar to F and W in geometric configuration, and contains a good hydrophobic side chain group, so W can be added to the Y similarity list. In amino acids, S contains a good side chain hydroxyl group, i.e. -OH. This amino acid only has commonality with H amino acid in LVLWGIHHP, i.e. it can act as an electron acceptor to produce polar hydrogen bonds.

[0117] Through the above sequence combination, a total of 2239488 LVLWGIHHP amino acid combinations and 3888 WSYIVE random combinations are obtained. Rank the sequence similarity of 3888 WSYIVE similar peptide segments and 2239488 LVLWGIHHP amino acids. By the method of sequence alignment intersection, it is found that almost all amino acids are included in PHHIGWLVL, so it is necessary to further narrow the range of protein amino acid sequence similarity.

[0118] Step D: Similarity amino acid matching. At least the physicochemical property is stronger than or the similarity amino acid replaces the amino acid in the polypeptide, improves the affinity of the combined fragment, or maintains the current affinity. Through the physicochemical property, the geometric fragment size (the size of the amino acid group volume) is differentiated more carefully by the similarity amino acid replacement. After layer-by-layer screening, the amino acids in the WSYIVE polypeptide randomly combined with the LVLWGIHHP randomly combined fragment have an overlap, and a total of 4608 predicted amino acids are obtained.

[0119] Step E: Scoring and ranking based on physicochemical properties and geometric fragment similarity for 4608 polypeptides, and finally obtaining 61 artificial combined polypeptides with the potential to bind to the active sites of 6 monoclonal antibodies.

[0120] 2) Screening and construction of recombinant multi-antigen epitope peptides that bind to each monoclonal antibody

[0121] First, 4608 polypeptides are randomly recombined by computer molecular simulation technology based on the key amino acids bound by each antibody, and then scored and ranked based on physicochemical properties and geometric fragment similarity, and finally 61 artificial recombinant multi-antigen epitope peptides with potential binding to the active sites of 6 monoclonal antibodies are obtained. 61 artificial recombinant polypeptides are used to dock proteins-polypeptides with Rosetta software, and the binding energy between 61 polypeptides and each monoclonal antibody is obtained. The average interaction energy between proteins and polypeptides includes electrostatic energy, van der Waals energy, and fuzzy interaction constraint energy. The binding energy between proteins and polypeptides is obtained and ranked comprehensively, and finally 6 polypeptides with good binding activity to monoclonal antibodies are ranked at the top, which are ILLWVLSHL, LLIWVLSHL, ILIWVLSHI, ILIWVLSHV, ILIWVISHL and ILIWVLSHL. Through biological experiments, their blocking effect, binding activity, and non-specificity of binding to antibodies are evaluated, and one multi-antigen epitope peptide is selected for further identification.

[0122] 3) Preparation and identification of immune serum

[0123] The recombinant multi-epitope peptide labeled with BSA was emulsified with Freund's complete adjuvant and subcutaneously immunized BALB / c mice, 30-50 μg per mouse; 4 weeks later, the recombinant multi-epitope peptide labeled with BSA was emulsified with Freund's incomplete adjuvant and immunized 2-3 times with the same dose as the initial immunization; two weeks apart, the last immunization was performed intraperitoneally with the antigen without adjuvant, and the tail vein blood was collected to prepare antisera. The indirect ELISA method was used, and the KLH-labeled recombinant multi-epitope peptide and H1N1 influenza virus antigen were coated, respectively, and washed after 5% skim milk powder was blocked at 4°C overnight. The obtained immune serum was used as the primary antibody, and the normal mouse serum was used as the control, and the secondary antibody was HRP-labeled goat anti-mouse secondary antibody (1:2500 dilution), which was operated according to the conventional indirect ELISA method. P / N>2.5 was taken as the positive result, so as to determine the titer of the immune serum.

[0124] Results

[0125] The single antibodies of the same short peptide have different reaction characteristics

[0126] In this study, the six single antibodies were reacted with 2009 H1N1 influenza virus split vaccine, H1N1, PR8, H3N2, H5N1, H7N2, H7N9 and other pathogens by ELISA, and the SP2 / 0 cell culture supernatant was used as a negative control. The specific results are shown in Table 2.

[0127] Table 2 Identification results of single antibodies of the same short peptide and different antigen reaction characteristics

[0128]

[0129] Note: The values in the table are the ratio of the ELISA detection OD value of the experimental group to the OD value of the SP2 / 0 control group (0.05 when the OD value of the SP2 / 0 control group is less than 0.05), and P / N≥2.5 is determined as positive; three duplicate wells are set in the experiment.

[0130] As can be seen from the results in Table 2, although A1-10, H1-74 and H1-84 recognize the same epitope peptide LVLWGIHHP, the reaction characteristics of the three single antibodies with antigens 501, H5N1 and H7N2 are not the same. In addition, the reaction of H1-5, H1-16 and H1-81 single antibodies recognizing epitope peptide WSYIVE with PR8 and H7N2 is also not the same, which indicates that the antigen epitopes they bind are not the same.

[0131] The key amino acid combinations of single antibodies recognizing the same short peptide are not the same

[0132] Two polypeptides (LVLWGIHHP, WSYIVE) were synthesized with alanine scanning substitution peptides, respectively; then the alanine scanning substitution peptides were respectively reacted with the above monoclonal antibodies, and the blocking effect of each peptide segment on the reaction of the monoclonal antibodies and HA antigen was observed. The blocking rate was calculated by measuring the OD value, and the key amino acid combinations of each monoclonal antibody binding to the polypeptide are shown in Table 3-A and B.

[0133] Table 3. Comparison of differences in key amino acid combinations of each monoclonal antibody binding

[0134]

[0135]

[0136] Note: The table shows which amino acids on the polypeptide play a key role in binding to the antibody when the polypeptide binds to different monoclonal antibodies. The experimental results were repeated 3 times (N = 3).

[0137] The results of Table 3 show that although A1-10, H1-74, and H1-84 recognize the polypeptide LVLWGIHHP, the key amino acids they recognize are not the same. In addition to V192, L193, W194, and I196 being the common skeleton recognized by the three monoclonal antibodies, A1-10 and H1-74 also recognize the key amino acid L191. The difference in the epitope they recognize lies in the fact that the former recognizes the key amino acid combination H197, while the latter recognizes P199. H1-84 recognizes a key amino acid combination that is one L191 less than H1-74, and the others are the same as H1-74 (see Table 3-A). H1-5, H1-16, and H1-81 bind to the polypeptide WSYIVE simultaneously, and each of them binds to four key amino acids. The four key amino acid combinations recognized by H1-16 and H1-81 differ by one amino acid, with I96 being used by one and V97 being used by the other. The other three key amino acids are the same. Of the four key amino acids bound by H1-5, only W93 is shared with the key amino acid combinations of the other two antibodies, I96 is shared with H1-16, and the other two key amino acids are not the same as those bound by the other two antibodies (Table 3-B). It can be seen that the six monoclonal antibodies each recognize six different key amino acid combinations, which may be the reason for the different reaction characteristics of the six monoclonal antibodies. At the same time, the amino acid composition of the light and heavy chain variable regions of the six monoclonal antibodies in Table 4 shows that the three monoclonal antibodies that bind to the same short peptide are not secreted by the same hybridoma cell, as shown in Table 4.

[0138] Table 4 Sequencing results of the heavy and light chain amino acid sequences of the six mAbs

[0139]

[0140]

[0141] Note: The amino acid composition of the light and heavy chain variable regions of the 6 monoclonal antibodies in Table 4 shows that the 3 monoclonal antibodies that bind to the same short peptide are not secreted by the same hybridoma cell.

[0142] Computer molecular simulation homology modeling of the 6 monoclonal antibodies and analysis of their interaction with 2 polypeptides

[0143] We further performed sequence alignment of the heavy chains and single chains of the 6 monoclonal antibodies and found that the RCSB PDB crystal database did not contain any protein structure with 100% sequence similarity. Therefore, we performed homology modeling of the protein structures of the monoclonal antibody systems (modeling templates are shown in Table 5). In homology modeling, the threshold for the similarity of the amino acid sequences of the proteins was 30%, and the higher the similarity, the higher the reliability of the modeled structure. Among them, the sequence similarity of the homology modeling templates of A1-10, H1-74, H1-84, H1-5, H1-16 and H1-81 monoclonal antibodies was all greater than 77%, and the modeling templates were highly reliable.

[0144] Table 5, Homology modeling of the protein structures of the monoclonal antibody systems

[0145]

[0146]

[0147] Note: The table shows how similar the templates used for modeling the variable regions of the 6 monoclonal antibodies are to the sequences of the monoclonal antibodies themselves, reflecting the reliability of the data obtained in subsequent simulation binding to polypeptides

[0148] Through Rosetta software, we performed protein-protein docking of the 6 monoclonal antibodies with two polypeptides (LVLWGIHHP and WSYIVE), respectively, and took the conformation with the lowest binding energy in the docking site of each system, and the interaction energy and the corresponding binding mode are shown in Table 6 and Figure 2The polypeptide LVLWGIHHP can form a dense network of binding interfaces with multiple amino acids on A1-10, H1-74 and H1-84 mAbs, and further form good polar interactions and hydrophobic binding. The binding energies between the three are not much different, floating in a small range of -20.7, -21.9, -22.8 kcal / mol, with good binding energies. The polypeptide WSYIVE is shorter than LVLWGIHHP, so the amino acids forming the binding interface between H1-5, H1-16 and H1-81 and the mAb are relatively less, but the polypeptide can still form good polar and hydrophobic binding matches with the binding pockets on each antibody, with energies of -14.63, -15.98 and -15.40 kcal / mol, respectively. The results of the bond energy analysis of the small molecule polypeptide and the antibody in Table 6 are basically consistent with the results of the key amino acid analysis of each antibody and the small molecule polypeptide WSYIVE in Table 3-B (the bond energy is the resultant force of the interaction between multiple amino acid residues on the antibody and one key amino acid on the polypeptide); each key amino acid on the small molecule polypeptide interacts with multiple amino acid residues on the variable region of the mAb.

[0149] Table 6A, Bond energy of each amino acid on the polypeptide WSYIVE and the amino acid residues on the variable regions of three mAbs

[0150]

[0151]

[0152] Note: The results in the table show that each amino acid on the antigenic epitope peptide can interact with multiple amino acid residues in the variable region of the antibody; and the same amino acid on the antigenic epitope peptide binds to different combinations of amino acid residues on the variable region of the antibody when binding to different antibodies.

[0153]

[0154] Note: The results in the table show that each amino acid on the antigenic epitope peptide can interact with multiple amino acid residues in the variable region of the antibody; and the same amino acid on the antigenic epitope peptide binds to different combinations of amino acid residues on the variable region of the antibody when binding to different antibodies.

[0155] 2 polypeptides LVLWGIHHP and WSYIVE exist in the antigenic epitope of influenza virus

[0156] By traditional hybridoma fusion and screening techniques, 16 and 10 strains of monoclonal antibodies were prepared against the two polypeptides LVLWGIHHP and WSYIVE respectively. After blocking experiments of the newly obtained monoclonal antibodies with the two polypeptides, there were only 7 strains of monoclonal antibodies whose binding activity could be completely blocked by the short peptides, respectively. The reaction characteristics of the 7 strains of monoclonal antibodies were analyzed, and it was found that among the newly prepared monoclonal antibodies against the two polypeptides, 1 and 7 strains of monoclonal antibodies could react with different subtypes of influenza virus antigens (see Tables 7 and 8), indicating that there were influenza virus antigen epitopes on the two polypeptides. Among the monoclonal antibodies against LVLWGIHHP, only LP-9-6 reacted with the antigens of influenza virus H1N1 and A3, indicating that the polypeptide at least composed two antigen epitopes, one of which was the same as a certain epitope on the antigens of influenza virus H1N1 and A3, and the other was different. Among the monoclonal antibodies against WSYIVE, although 7 strains of monoclonal antibodies all reacted with certain subtypes of influenza virus antigens, the reaction characteristics of the other 4 strains of monoclonal antibodies were different from each other except for the three strains of monoclonal antibodies WE-6-11, WE-6-12 and WE-6-16, suggesting that the polypeptide composed multiple antigen epitopes.

[0157] Table 7 Reaction characteristics identification of monoclonal antibodies against LVLWGIHHP with immunogens and influenza virus antigens

[0158]

[0159] Note: The values in the table are the ratio of the OD values of the experimental group to the OD values of the SP2 / 0 control group (antibodies and SP2 / 0 negative control use cell culture supernatant, and when the OD value of the negative control is less than 0.05, it is counted as 0.05), P / N≥2.5 is determined as positive; three duplicate wells are set in the experiment.

[0160] Table 8 Reaction characteristics identification of monoclonal antibodies against WSYIVE with immunogens and influenza virus antigens

[0161]

[0162] Note: The values in the table are the ratio of the OD values of the experimental group to the OD values of the SP2 / 0 control group (antibodies and SP2 / 0 negative control use cell culture supernatant, and when the OD value of the negative control is less than 0.05, it is counted as 0.05), P / N≥2.5 is determined as positive; three duplicate wells are set in the experiment.

[0163] Recombinant polypeptide ILLWVLSHL induced immune response against influenza virus HA antigen obtained by computer molecular simulation technology

[0164] We recombine the key amino acids of 6 monoclonal antibodies to obtain 61 recombinant multi-antigen epitope peptides that may contain the epitopes of 6 monoclonal antibodies. We obtain the energy ranking of 61 artificial recombinant polypeptides and 6 monoclonal antibody proteins by molecular docking method, so as to obtain 6 recombinant multi-antigen epitope peptides with an affinity greater than -8.0 Kcal / mol to each monoclonal antibody. The results are shown in Table 9. As can be seen from the table, the energy scores of 6 monoclonal antibodies and recombinant polypeptides ILLWVLSHL, LLIWVLSHL, ILIWVLSHI, ILIWVLSHV, ILIWVISHL, and ILIWVLSHL are significantly reduced, indicating that the 6 recombinant polypeptides have potential activity for simultaneous binding with 6 monoclonal antibodies, and that the 6 recombinant multi-antigen epitope peptides carry the target antigen epitopes of 6 monoclonal antibodies and can be used for subsequent analysis.

[0165] Table 9 Sequences of 6 recombinant multi-antigen epitope peptides recommended by computer and affinity of binding with antibodies

[0166]

[0167] We perform blocking experiments of 6 recombinant multi-antigen epitope peptides on the binding of 6 monoclonal antibodies and influenza virus antigens, and the results are shown in Figure 3 As shown in the table, the inhibition rates of multi-antigen epitope peptides XH-001: ILLWVLSHL and XH-006: ILIWVLSHL on the binding of 6 monoclonal antibodies and antigens are all above 0.8; the inhibition rates of other polypeptides on the binding of 6 monoclonal antibodies and antigens are all below 0.4 or between 0.4 and 0.8; the multi-antigen epitope peptides ILLWVLSHL and ILIWVLSHL can simultaneously block the binding of 6 monoclonal antibodies and influenza virus antigens, indicating that the multi-antigen epitope peptides carry antigen epitopes that can bind well with 6 antibodies.

[0168] In order to exclude non-specific reactions in the experiment, we analyze the specific reactions of 2 recombinant multi-antigen epitope peptides screened by blocking experiments and the cross-reactions of irrelevant monoclonal antibodies, and the results show that the antigen epitopes of 6 monoclonal antibodies bound to multi-antigen epitope peptide XH-001 ILLWVLSHL are well preserved after labeling the carrier protein (see Table 10), while the epitopes bound by monoclonal antibodies H1-74, H1-84, H1-5, and H1-81 on multi-antigen epitope peptide XH-006 are lost in some steps of the experiment. Therefore, we select multi-antigen epitope peptide XH-001 for the next animal immunization experiment.

[0169] Table 10 Specific reactions of recombinant multi-antigen epitope peptides and 6 monoclonal antibodies and cross-reactions of irrelevant monoclonal antibodies

[0170]

[0171] Note: The values ​​in the table are the ratios of the OD values ​​of the experimental group detected by ELISA to those of the SP2 / 0 control group (if the SP2 / 0 OD value is less than 0.05, it is counted as 0.05). P / N ≥ 2.5 is considered positive. The experiment was set up with 3 replicates (N = 3).

[0172] Balb / c mice were routinely immunized with a polyantigen epitope peptide labeled with BSA, and the production of antibodies against two small short peptides, LVLWGIHHP, WSYIVE, and H1N1 influenza virus was detected. The results in Table 11 show that although the amino acid sequence of the recombinant polyantigen epitope peptide ILLWVLSHL is different from that of the two small peptides, the experimental results of its immune serum reacting with both small peptides and influenza virus antigens indicate that the recombinant polyantigen epitope peptide screened using six monoclonal antibodies as templates not only still carries the antigenic epitopes of the two small peptides, but also that the antigenic epitopes on the influenza virus HA protein were well replicated onto the recombinant polyantigen epitope peptide by the six anti-influenza virus monoclonal antibodies, and these epitopes were well displayed when immunizing animals.

[0173] Table 11. ELISA results of antibodies against the target antigenic epitope in the serum of mice immunized with recombinant multiantigen epitope peptides.

[0174]

[0175] Note: The values ​​in the table represent the values ​​of four B12 mice immunized with the recombinant multiantigen epitope peptide vector conjugate XH001-BSA. a lB / c Mouse-derived anti-natural peptide LVLWGIHHP,

[0176] The antibody titers of WSYIVE, recombinant multiantigen epitope peptide ILLWVLSHL, and H1N1 influenza virus antigen were measured. A positive result was defined as an OD value greater than 2.5 in the experimental group compared to the OD value of the normal mouse serum control group (normal mouse serum OD value less than 0.05 was considered as 0.05). The experiment was repeated 3 times (N=3).

[0177] Based on previous research, this invention has discovered that several monoclonal antibodies against the influenza virus HA protein are located on the same peptide segment.

[10] However, the amino acid sequences of the variable regions of these monoclonal antibodies were not identical, indicating that these monoclonal antibodies were not secreted by a single hybridoma cell (Table 4); and the reaction characteristics of the three monoclonal antibodies binding to the same peptide segment were not identical, suggesting that the three monoclonal antibodies might not bind to the same antigenic epitope on the same short peptide (Table 2); that is, the peptide segment might have formed more than one antigenic epitope. Analysis of the binding epitopes of the antibodies by the alanine scanning amino acid substitution method showed that there were 1-2 amino acid differences between the key amino acid combinations bound by the antibodies (Table 3), suggesting that the reason for the different reaction characteristics of the three monoclonal antibodies binding to the same short peptide might be the difference in the key amino acid combinations bound by the antibodies; that is, the antibodies might have been stimulated by different key amino acid combinations. This result indicates that the basic functional unit of a polypeptide antigen, an antigenic epitope, might be a combination of key amino acids.

[0178] To verify whether a small molecule short peptide can form multiple antigenic epitopes, we prepared monoclonal antibodies from two peptide segments, LVLWGIHHP and WSYIVE, from the HA protein of an influenza virus; the results showed that the monoclonal antibodies stimulated by the two peptide segments could or could not bind to the HA antigen, and the reaction characteristics of the monoclonal antibodies that bound to the HA antigen were not identical to those that did not bind to the HA antigen, suggesting that a small molecule 6-peptide might have formed multiple different antigenic epitopes (Tables 7 and 8). These results suggest that an antigenic epitope might not be a fixed structure, but a combination of multiple key amino acids (or certain groups thereon); if so, the groups in the key amino acid combination forming an antigenic epitope might be replaced by groups with the same or similar properties; that is, the key amino acid combination forming an antigenic epitope might be split and recombined. This characteristic provides a theoretical basis for designing antibodies with different reaction characteristics.

[0179] In this subject, at least three or more monoclonal antibody binding epitopes were concentrated on such a short peptide segment, indicating the complexity of the composition of an antigenic epitope. The above results show that the binding of a small molecule polypeptide to an antibody is actually the interaction between the amino acids (groups) on the polypeptide chain and the groups on the amino acid residues of the variable region of the antibody; whether they can bind depends on their surface accessibility and affinity; if these conditions are met, the groups playing an immunodominant role in the polypeptide can bind to different antibodies in different combinations (Tables 6 and 9). That is, multiple key amino acids in a polypeptide can play an immunostimulatory role as different antigenic epitopes in different combinations; which or which combination of these key amino acids plays an immunostimulatory role depends on the selection of immune cells (Tables 2, 3, and 4).

[0180] Based on the above, the present study proposes that the polypeptide epitope is a combination of key amino acids, and these key amino acids can be replaced by key amino acids of the same nature, and multiple antigen epitopes can be combined and recombined into a multi-antigen epitope peptide. To verify the correctness of this point of view, the present study uses computer simulation technology to synthesize multi-antigen epitope polypeptides that are non-homologous to the original protein for antigen epitope prediction, combination and recombination, or application. The present study uses computer molecular simulation technology to split the key amino acid combination of 6 monoclonal antibodies and recombine them into a multi-antigen epitope peptide (attached Figure 1 ). The variable region of the 6 monoclonal antibodies is subjected to protein structure homology modeling (Table 5), and the binding activity of the recombinant multi-antigen epitope peptide to the 6 monoclonal antibodies is analyzed under the guidance of computer molecular simulation technology (Table 6, Figure 2 ). It is proved that although the sequence of the recombinant multi-antigen epitope peptide is different from that of the polypeptides LVLWGIHHP and WSYIVE, the recombinant multi-antigen epitope peptide indeed carries antigen epitopes that can bind to the 6 monoclonal antibodies (Tables 7, 8, 9, 10, Figure 3 ). After mice were routinely immunized with BSA-labeled recombinant multi-antigen epitope peptide ILLWVLSHL, strong antibodies against polypeptides LVLWGIHHP, WSYIVE and H1N1 influenza virus antigen were induced (Table 11). This proves that the antigen epitopes on the two polypeptides and the H1N1 antigen HA protein were transferred to the recombinant multi-antigen epitope peptide using the 6 monoclonal antibodies as a template, that is, the multi-antigen epitope peptide contains similar antigen epitopes to the two polypeptides and the H1N1 influenza virus antigen. The slightly higher antibody titer of the recombinant multi-antigen epitope peptide immune serum against the influenza virus H1N1 antigen than that of the anti-polypeptide LVLWGIHHP, WSYIVE antibody titer seems to suggest that the recombinant multi-antigen epitope peptide ILLWVLSHL carries more identical antigen epitopes to the influenza virus H1N1 antigen than the two polypeptides LVLWGIHHP and WSYIVE each carrying identical antigen epitopes. Thus, we establish a method of multi-antigen epitope combination and recombination under the guidance of computer molecular simulation technology, and use the variable region of the antibody as a template to screen the multi-antigen epitope. Moreover, it is proved that the recombinant peptide carrying the multi-antigen epitope can also well display its immunogenicity when immunized.

[0181] The present study proposes the concept that polypeptide epitopes are combinations of key amino acids. According to the concept, an epitope is a combination of key amino acids, and the groups in the combination can come from adjacent amino acids or from amino acids that are not adjacent and are far apart in the amino acid sequence of a protein. These key amino acids, which are gathered together, can bind to different immune cells in different combinations to exert immune stimulation. Which combination of immunodominant groups exerts immune stimulation depends entirely on the selection of antigen receptors on immune cells. Perhaps we can try to enrich our understanding of antigen epitopes by establishing a database by matching antibodies to key amino acids of antigens.

[0182] On the other hand, we synthesized non-influenza virus polypeptides that can induce the production of anti-influenza virus HA antibodies through computer molecular simulation technology. The establishment and practice of this new concept provide new theoretical and applied research ideas for the research and development of polypeptide vaccines, multivalent vaccines and polypeptide drugs. Our concept of single polypeptide loading multiple epitopes will be beneficial to the design and application of new coronal vaccines. In addition, our method can also be used for other researches. For example, in the research of autoimmune diseases, we will abandon the previous attempts to find corresponding relationships between existing antigen epitope libraries and human self-antigen epitopes, and use the concept of immunodominant group combinations to find antigen epitopes that are different in structure or sequence but have similar antibody functions. In addition, our method can also be used in tumor-related (specific) development, confirmation of polypeptide-related new diagnostic epitopes and polypeptide drug research and development.

[0183] Example 2 Application of the screening method in the preparation of polypeptide vaccines, multivalent vaccines or polypeptide drugs

[0184] In view of the variability of the new coronavirus, the key amino acid combinations used by the five subtypes of Alpha, Beta, Gamma, Delta and Omicron viruses to bind to ACE2 are split and recombined into many multi-antigen epitope peptides under the guidance of computer molecular simulation technology. Then, the ACE2 receptor protein crystal model combined with each multi-antigen epitope peptide is simulated to screen various polypeptides that can bind to the ACE2 receptor. The polypeptides are labeled with a carrier, and mice are immunized. The immune serum is detected by biological neutralization test to determine the serum with high titer that can block the binding of the five main subtypes of viruses to ACE2. The multi-antigen epitope peptide used to prepare the serum is used as a new coronavirus vaccine, and a multivalent vaccine with strong antigenicity against the five subtypes of new coronaviruses can be developed.

[0185] For the new coronavirus vaccine, HIV vaccine and other vaccines with weak immunogenicity, it is difficult to stimulate the inoculated person to produce neutralizing antibodies after vaccination, or the neutralizing antibodies produced are short-lived. Natural vaccines can be recombined. For example, 16 amino acids on the S protein spike of the new coronavirus S protein combined with the ACE2 receptor protein, sequence recombination under the guidance of computer molecular simulation technology, then simulate the combination of ACE2 receptor protein with recombinant polypeptide, screen out recombinant polypeptide with good combination with receptor protein, then label the recombinant polypeptide with a carrier, immunize animals routinely, detect the titer of neutralizing antibodies against the new coronavirus in immune serum, and select the recombinant polypeptide that can stimulate high-titer neutralizing antibodies to prepare vaccine.

Claims

1. A method for multiple antigenic epitope convergent recombination based on computer molecular modeling techniques, characterized in that, The method comprises the following steps: S1, determine the number of monoclonal antibody binding epitopes to be combined and recombined, and analyze the key amino acid combination of each monoclonal antibody binding epitope; In S1, the key amino acid combination of each monoclonal antibody binding epitope is analyzed by replacing the amino acids in the polypeptide with alanine one by one, then synthesizing the alanine replacement peptide, and detecting the influence of the replaced amino acids in the polypeptide on the binding of the polypeptide and the monoclonal antibody by ELISA blocking method; if the replaced amino acids have a great influence on the binding of the polypeptide and the antibody, it indicates that the replaced amino acids play a great role in the binding of the polypeptide and the antibody, and are the key amino acids; otherwise, they are non-key amino acids; S2, under the guidance of computer molecular simulation technology, all key amino acids of the planned combined antigen epitopes are combined and recombined to form a multi-antigen epitope peptide containing multiple monoclonal antibody binding epitopes; In S2, thousands of multi-antigen epitope peptides are randomly recombined from the key amino acids of each antibody binding according to the size of the recombined peptides by computer molecular simulation technology, and ranking is performed based on the physicochemical properties of the polypeptides and the geometric fragment similarity; S3, retrieve the mRNA of the monoclonal antibody hybridoma cell strain, synthesize cDNA by reverse transcription, amplify the light chain variable region gene and the heavy chain variable region gene of each monoclonal antibody respectively, and obtain the amino acid sequence of the monoclonal antibody variable region; construct a three-dimensional structure model of the monoclonal antibody variable region by computer molecular simulation technology; In S3, the method for constructing a three-dimensional structure model of the monoclonal antibody variable region by computer molecular simulation technology is as follows: selecting crystal proteins with good similarity to the amino acid sequences of each monoclonal antibody from RCSB PDB protein crystal database as templates for homology modeling, and using homology modeling software Modeller to construct a three-dimensional structure model of the variable region of each monoclonal antibody; S4, each monoclonal antibody is combined with each recombined multi-antigen epitope peptide by computer molecular simulation technology to simulate the binding; ranking the binding energy between the recombined multi-antigen epitope peptide and each monoclonal antibody obtained by molecular simulation combination, and screening and recommending the recombined multi-antigen epitope peptide sequence that can be well combined with each monoclonal antibody; In S4, UCSF Chimera software is used to construct the three-dimensional structure of each monoclonal antibody target antigen peptide; Rosetta software is used to dock each monoclonal antibody with the top-ranked multi-antigen epitope peptide in S2, and polypeptide synthesis and biological verification are performed on the polypeptide with good binding activity and the top-ranked polypeptide for each monoclonal antibody; S5, polypeptide synthesis and biological identification are performed on the screened and recommended multi-antigen epitope peptide to determine whether it meets the design requirements; In S5, the design requirements include but are not limited to the biological indicators required by the design purposes such as the binding activity of the recombined multi-antigen epitope peptide with each monoclonal antibody, the blocking effect on the binding of the antibody and the target antigen, and the cross-reactivity with the antibody, etc. laboratory evaluation, and the multi-antigen epitope peptide with good evaluation results and meeting the design purposes is screened out for further research or application.

2. The method of multi-antigenic epitope amalgamation recombination based on computer molecular simulation technology according to claim 1, characterized in that, The S2, by computer molecular simulation technology to plan the key amino acids of the combined antigen epitope are split, combined, recombined steps include: Step A: using Cd-hit software to each single antibody binding antigen epitope peptide key amino acid sequence similarity matching; Step B: according to the amino acid geometry and physicochemical properties, the single antibody binding antigen epitope peptide for global physicochemical property matching; Step C: geometric configuration similarity matching; Step D: similarity amino acid matching; Step E: to random recombination of multiple antigen epitope peptide, based on physicochemical properties and geometric fragment similarity, scoring ranking, finally get dozens or hundreds of multiple antigen epitope peptide with the potential to bind multiple single antibody active site.

3. A method for obtaining a synthetic recombinant polyepitope peptide, characterized in that, The method for recombining multiple antigen epitopes based on computer molecular simulation technology according to claim 1 or 2 is used to realize the single antibody of the target pathogenic microorganism protein with different reaction characteristics and the polypeptide combined with the single antibody as the research object, under the guidance of computer molecular simulation technology, the key amino acid combination of the single antibody combined with the target pathogenic microorganism antigen polypeptide is split, combined and recombined, and the recombined multiple antigen epitope peptide is screened with the single antibody variable region as the template, and the artificial recombined multiple antigen epitope peptide is obtained.

4. The recombined multiple antigen epitope peptide with the function of inducing anti-influenza HA antigen immune response, and the sequence is ILLWVLSHL.

5. The application of the recombined multiple antigen epitope peptide with the function of inducing anti-influenza HA antigen immune response in claim 4 in the preparation of polypeptide vaccine.

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