Conjugated protein monomer carrying peptide derived from pathogenic microorganism compatible with MHC molecule, aggregate of said monomers, component vaccine containing said aggregate as active ingredient, and method for acquiring information on secretion of physiologically active substance after immunization

Abstract

The present invention addresses the problem of establishing a means for providing a component vaccine that can achieve both of the induction of cellular immunity mainly associated with MHC class I and the induction of humoral immunity associated with MHC class II selectively or intensively. The present inventors have found that the problem can be solved by providing a component vaccine which contains, as an active ingredient, aggregates each comprising a trimer and / or a hexamer of a molecular needle carrying a peptide capable of binding to MHC class I and / or a peptide capable of binding to MHC class II. The present inventors have also discovered an information acquisition method, in which the change in secretion of a physiologically active substance, e.g., cytokine, in an animal of interest after the infection with a target microorganism is detected to evaluate an attribute of MHC class, e.g., a peptide of interest or the like.

Description

A composite protein monomer carrying a peptide derived from a pathogenic microorganism that matches an MHC molecule, an aggregate of the monomer, a component vaccine containing the aggregate as an active ingredient, and a method for obtaining information regarding the secretion of physiologically active substances after immunization.

[0001] The present invention relates to functional proteins and vaccines using the same. More specifically, it relates to an immunogen, a "composite protein carrying a peptide derived from a pathogenic microorganism such as a virus or bacterium that matches an MHC molecule" (monomer), an aggregate of the monomer (molecular needle), and a component vaccine that uses the aggregate as an active ingredient (protective antigen). Furthermore, it relates to a method for obtaining information on the secretion of physiologically active substances after immunization from a peptide or protein that is an active ingredient of the vaccine.

[0002] A technology for a "molecular needle" (Patent Document 1) has been proposed, which was invented by focusing on the excellent gene transfer function of bacteriophages into cells.

[0003] The present inventors have invented a composite protein in which a structural protein of norovirus is carried on this molecular needle, and have provided it as a component vaccine against norovirus, and have filed a patent application for this (Patent Document 2).

[0004] JP 2015-163056 A, WO2018 / 074558 International Publication Pamphlet

[0005] Hoffmann et al.,2020,Cell 181,1-10 April 16,2020Daniel Wrapp et al., Science, published online February 2020, science abb2507

[0006] "Disease enhancement" is a very serious problem when developing a vaccine. "Disease enhancement" is a phenomenon in which antibodies obtained through infectious disease prevention vaccination enhance subsequent infection or inflammation that occurs after infection, and the mechanism by which this occurs has not yet been elucidated. Disease enhancement that may be caused by COVID-19 (SARS-CoV-2) vaccination is called antibody-dependent enhancement of disease (ADE) or enhanced respiratory disease (ERD). In addition, in the development of vaccines for betacoronaviruses, the possibility of disease enhancement has been reported in animal experiments of vaccines for severe acute respiratory syndrome (SARS) virus and Middle East respiratory syndrome (MERS) virus (Honda-Okubo Y, et al., J Virol. 2015;89(6):2995-3007, Agrawal AS, et al., Hum Vaccin Immunother. 2016;12(9):2351-56). Furthermore, in clinical trials of vaccines for respiratory syncytial virus (RSV) and dengue fever virus, as well as coronavirus vaccines, deaths and severe illnesses possibly related to infection enhancement have been reported (Castilow EM, et al., Immunol Res. 2007;39(1-3):225-39, Halstead SB. Hum Vaccin Immunother. 2018;14(9):2158-62).

[0007] Thus, vaccination-induced disease enhancement is one of the obstacles to vaccine development.

[0008] The present inventors conducted research to solve the above problems using a component vaccine using a molecular needle. Surprisingly, they discovered that by using a molecular needle carrying a peptide derived from a pathogenic microorganism that matches an MHC molecule as an active ingredient, it is possible to select either cellular or humoral immunity induced by vaccination, or to adjust the balance between the two types of immunity, and to efficiently improve the protective effect against pathogenic microorganisms. This finding led to the completion of the present invention. This makes it possible to avoid the problem of "vaccination-associated disease enhancement." Furthermore, it makes it possible to induce the desired immunity extremely efficiently and accurately. Furthermore, as a booster vaccine, it can selectively enhance the immune response required for memory immunity induced by the primary vaccine.

[0009] The vaccine of the present invention is a component vaccine containing, as an active ingredient, a molecular needle carrying a peptide derived from a pathogenic microorganism compatible with MHC class I and / or a peptide derived from a pathogenic microorganism compatible with MHC class II.

[0010] That is, the peptide carried by the molecular needle, which is the active ingredient of the vaccine of the present invention, includes three types: (1) a peptide derived from a pathogenic microorganism compatible with MHC class I; (2) a peptide derived from a pathogenic microorganism compatible with MHC class II; and (3) a peptide derived from a pathogenic microorganism compatible with MHC class I and a peptide derived from a pathogenic microorganism compatible with MHC class II.

[0011] In other words, the first vaccine of the present invention is a component vaccine (vaccine 1 of the present invention) containing, as an active ingredient, a molecular needle carrying a peptide derived from a pathogenic microorganism that matches MHC class I; the second vaccine of the present invention is a component vaccine (vaccine 2 of the present invention) containing, as an active ingredient, a molecular needle carrying a peptide derived from a pathogenic microorganism that matches MHC class II; and the third vaccine of the present invention is a component vaccine (vaccine 3 of the present invention) containing, as an active ingredient, a molecular needle carrying a peptide that functions as both a peptide derived from a pathogenic microorganism that matches MHC class I and a peptide derived from a pathogenic microorganism that matches MHC class II.

[0012] "Compatible with MHC class I" means that it promotes a specific function in the body's immune system by MHC class I molecules (described below: in short, the induction of cellular immunity), and "compatible with MHC class II" means that it promotes a specific function in the body's immune system by MHC class II molecules (described below: in short, the preferential induction of humoral immunity).

[0013] Vaccine 1 of the present invention can selectively induce cellular immunity against a target pathogenic microorganism. Vaccine 2 of the present invention can preferentially induce humoral immunity against a target pathogenic microorganism. Vaccines 1 and 2 of the present invention can be used in combination or alone, even if they target the same pathogenic microorganism. Such combined or individual use can achieve a balance between eliciting cellular and humoral immunity and efficiently improve the protective effect against pathogenic microorganisms. For example, disease enhancement associated with vaccination is often thought to be caused by runaway humoral immunity. Using vaccine 1 of the present invention alone or reducing the ratio of vaccine 2 of the present invention in combination can prevent such disease enhancement. Conversely, disease enhancement caused by runaway cellular immunity is also known. In such cases, it is preferable to use vaccine 2 of the present invention, which contains a molecular needle carrying an MHC class II peptide as an active ingredient, in contrast to the above. Furthermore, by selecting aggregates 1 and 2 of the present invention so as to actively exert both cellular and humoral immunity and combining them as the active ingredient of a vaccine, it is possible to efficiently design a vaccine that is highly effective in protecting against pathogenic microorganisms. Vaccine 3 of the present invention is a component vaccine that models the balance between cellular and humoral immunity or effectively exerts the effects of both, by carrying a molecular needle that is used as the active ingredient of a component vaccine, and is composed of a peptide complex formed by predetermining the combination and ratio of a peptide derived from a pathogenic microorganism that matches MHC class I and a peptide derived from a pathogenic microorganism that matches MHC class II, or a peptide that functions as both a peptide derived from a pathogenic microorganism that matches MHC class I and a peptide derived from a pathogenic microorganism that matches MHC class II (preferable examples of such a peptide that functions in both ways include a peptide sequence that binds to a B cell receptor in the humoral immune pathway involving MHC class II).

[0014] Thus, the vaccine of the present invention conceptually encompasses vaccines 1, 2, and 3 of the present invention.

[0015] The molecular needle that serves as the active ingredient of vaccine 1 of the present invention is an aggregate of complex protein 1 of the present invention (aggregate 1 of the present invention) described below; the molecular needle that serves as the active ingredient of vaccine 2 of the present invention is an aggregate of complex protein 2 of the present invention (aggregate 2 of the present invention) described below; and the molecular needle that serves as the active ingredient of vaccine 3 of the present invention is an aggregate of complex protein 3 of the present invention (aggregate 3 of the present invention) described below.

[0016] [1] The conjugated protein of the present invention is a conjugated protein having an amino acid sequence of the following formula (1): １ -X ｎ -Y (1) [wherein W represents one or more amino acid sequences of peptides containing one or more of the immunogens, which are peptides derived from pathogenic microorganisms compatible with MHC class I and / or peptides derived from pathogenic microorganisms compatible with MHC class II, and L １ represents a first linker sequence having 0-100 amino acids, X represents the amino acid sequence of SEQ ID NO: 1, Y represents the amino acid sequence of the cell introduction region, and n, which is the number of repetitions of X, is an integer of 1-10.], and the amino acid sequence of the cell introduction region Y is represented by the following formula (2): Y １ -L ２ -Y ２ -Y ３ (2) [wherein, Y １ represents any one amino acid sequence selected from the group consisting of SEQ ID NOs: 2-5, and Y ２ represents any one amino acid sequence selected from the group consisting of SEQ ID NOs: 6-9, and L ２ represents a second linker sequence of 0-30 amino acids, and Y ３ indicates the amino acid sequence for modification, and Y ２ or Y ３ In some cases, X may not be present.] ｎ , Y １ , or Y ２ It is also a composite protein which is allowed to contain a modified amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in the formula (I).

[0017] In the formulas (1) and (2), the "-" connecting the predetermined amino acid sequences represents W, L, １ , X ｎ , Y, etc., in order to clearly distinguish between conceptually grouped amino acid sequences, it is a representation of a simple molecular bond (essentially a peptide bond).

[0018] Above X ｎ , Y １ , or Y ２ "Deletion" in the amino acid sequence represented by SEQ ID NO: means that any amino acid residue in the amino acid sequence of each SEQ ID NO: defined in the above formula is deleted, and the amino acid residues on the N-terminal side (before) and C-terminal side (after) of the deleted amino acid residue are linked by a peptide bond (in the case of deletion of the N-terminal amino acid residue and the C-terminal amino acid residue, the amino acid residue is simply deleted), and the number of deleted residues is counted as the "number of amino acid deletions." "Substitution" means that any amino acid residue in the amino acid sequence of each SEQ ID NO: defined in the above formula is replaced with "another amino acid residue," and the replaced amino acid residue is linked by a peptide bond to each amino acid residue on the N-terminal side (before) and C-terminal side (after) (in the case of substitution of the N-terminal amino acid residue, only the peptide bond with the C-terminal amino acid residue is formed, and in the case of substitution of the C-terminal amino acid residue, only the peptide bond with the N-terminal amino acid residue is formed), and the number of substituted residues is counted as the "number of amino acid substitutions." "Addition" refers to the formation of new peptide bonds by inserting one or more new amino acid residues at any one or more peptide bond positions in the amino acid sequence of each SEQ ID NO: defined in the above formula. The nature and number of these amino acid residue modifications can be determined by aligning the amino acid sequence according to the above formula with the amino acid sequence of the modifications manually or on a computer using software capable of analyzing amino acid sequences.

[0019] Also, the linker sequence L defined by the above formula １ or L ２ , or the amino acid sequence Y for modification ３can be selected as needed within the range of the number of amino acid residues defined above.

[0020] It is preferable that the trimer or hexamer of the modified composite protein having the modified amino acid sequence has substantially the same immunostimulatory activity as the trimer or hexamer of the composite protein having the above formula. "Substantially equivalent" means a degree of equivalence in which, when an established method for confirming immunostimulatory activity, such as a "neutralization test," is used, no significant difference in immunostimulatory activity between the modified composite protein and the unmodified composite protein having the amino acid sequence is observed at a significance level of 5% or less.

[0021] In the above formula, X ｎ , Y １ , or Y ２ In the amino acid sequences represented by the formula (I), the number of amino acid modifications in each amino acid sequence in which one or more amino acids are deleted, substituted, or added is X ｎ is 8n or less, preferably 4n or less, more preferably 2n or less, most preferably 1n or less; Y １ are 30 or less, preferably 20 or less, more preferably 10 or less, extremely preferably 2 or less, and most preferably 1 or less; and Y ２ is 15 or less, preferably 10 or less, more preferably 5 or less, extremely preferably 2 or less, and most preferably 1 or less. These deletions, substitutions, or additions of amino acids are permitted on the premise that the conjugated proteins 1, 2, and 3 of the present invention form trimers or hexamers, respectively.

[0022] Complex protein 1 of the present invention is an embodiment of the above-mentioned complex protein of the present invention in which W contains only a peptide derived from a pathogenic microorganism that matches the immunogen MHC class I; complex protein 2 of the present invention is an embodiment in which W contains only a peptide derived from a pathogenic microorganism that matches the immunogen MHC class II; and complex protein 3 of the present invention is an embodiment in which W contains both a peptide derived from a pathogenic microorganism that matches the immunogen MHC class I and a peptide derived from a pathogenic microorganism that matches the immunogen MHC class II.

[0023] In the conjugated protein of the present invention, a preferred form of W is a peptide in which two or more peptides derived from pathogenic microorganisms matching MHC class I and / or two or more peptides derived from pathogenic microorganisms matching MHC class II are linked via a linker. As shown in the Examples, a preferred form of W is a peptide containing both a peptide portion (motif) matching MHC class I and a peptide portion (motif) matching class II. Furthermore, a preferred form of W is a peptide derived from a pathogenic microorganism that can be recognized by a B cell receptor.

[0024] The present invention also provides a gene expression vector (hereinafter also referred to as the vector of the present invention) incorporating a nucleic acid encoding the complex protein of the present invention, and further provides a transformant (hereinafter also referred to as the transformant of the present invention) transformed with a nucleic acid encoding the complex protein of the present invention. The vector of the present invention also includes an embodiment in which a nucleic acid encoding complex protein 1 of the present invention is incorporated (vector 1 of the present invention), an embodiment in which a nucleic acid encoding complex protein 2 of the present invention is incorporated (vector 2 of the present invention), and an embodiment in which a nucleic acid encoding complex protein 3 of the present invention is incorporated (vector 3 of the present invention).

[0025] The transformant of the present invention can be easily obtained by transforming a host with the vector of the present invention. The transformant of the present invention also includes an embodiment transformed with vector 1 of the present invention (transformant 1 of the present invention), an embodiment transformed with vector 2 of the present invention (transformant 2 of the present invention), and an embodiment transformed with vector 3 of the present invention (transformant 3 of the present invention).

[0026] The conjugated protein of the present invention can be produced by expressing genes in the transformant of the present invention.

[0027] [2] Associate of the Present Invention (2-1) Associate of the Present Invention Associate of the Present Invention, which is the active ingredient of vaccine 1 of the present invention, is an aggregate in which complex protein 1 of the present invention is a monomer, and contains a trimer or hexamer of complex protein 1, or a mixture of the trimer and hexamer. Hereinafter, the substance contained in aggregate 1 of the present invention may be collectively referred to as "trimer and / or hexamer 1." In other words, aggregate 1 of the present invention is an aggregate containing trimers and / or hexamers in which complex protein 1 of the present invention is a monomer. Furthermore, in consideration of the production process of complex 1 of the present invention described below, aggregate 1 of the present invention can also be defined as an aggregate formed by associating complex protein 1 of the present invention in an aqueous liquid. Associate 1 of the present invention is capable of penetrating into cells by itself.

[0028] The trimer is a trimeric protein composed of the same or different complex proteins 1 of the present invention as monomer proteins, and the hexamer is a hexamer protein composed of two molecules of the trimeric protein 1 associated with each other.

[0029] The aggregate 1 of the present invention can be produced by contacting the conjugated protein 1 of the present invention in an aqueous liquid. Depending on the compatibility between the peptide to be supported and the molecular needle, the aggregate 1 may not be formed, or even if an aggregate is formed, it may not be soluble in the aqueous liquid. However, by contacting the target conjugated protein in an aqueous liquid and confirming the results using SDS-PAGE, high-speed atomic force microscopy, gel filtration chromatography, or the like, it is possible to easily determine whether the target conjugated protein forms a trimer or hexamer and can be used as the active ingredient of the vaccine 1 of the present invention. Furthermore, solubility in an aqueous liquid can be easily confirmed by actually conducting a solubilization test.

[0030] (2-2) Associate 2 of the Present Invention Associate 2 of the present invention, which is the active ingredient of vaccine 2 of the present invention, is an aggregate in which complex protein 2 of the present invention is a monomer, and contains a trimer or hexamer of complex protein 2, or a mixture of the trimer and hexamer. Hereinafter, the substance contained in associate 2 of the present invention may be collectively referred to as "trimer and / or hexamer 2." In other words, associate 2 of the present invention is an aggregate containing trimers and / or hexamers in which complex protein 2 of the present invention is a monomer. Furthermore, in consideration of the production process of complex 2 of the present invention described below, associate 2 of the present invention can also be defined as an aggregate formed by associating complex protein 2 of the present invention in an aqueous liquid. Associate 2 of the present invention is capable of penetrating into cells by itself.

[0031] The trimer is a trimeric protein composed of the same or different complex proteins 2 of the present invention as monomer proteins, and the hexamer is a hexameric protein composed of two molecules of the trimeric protein 2 associated with each other.

[0032] The aggregate 2 of the present invention can be produced by contacting the conjugated protein 2 of the present invention in an aqueous liquid. Depending on the compatibility between the peptide to be supported and the molecular needle, the above-mentioned aggregate may not be formed, or even if an aggregate is formed, it may not be soluble in the aqueous liquid. However, by contacting the target conjugated protein 2 in an aqueous liquid and confirming the results using SDS-PAGE, high-speed atomic force microscopy, gel filtration chromatography, or the like, it is possible to easily determine whether the target conjugated protein forms a trimer or hexamer and can be used as an active ingredient in the vaccine 2 of the present invention. Furthermore, solubility in an aqueous liquid can be easily confirmed by actually conducting a solubilization test.

[0033] (2-3) Associate 3 of the Present Invention The associate 3 of the present invention, which is the active ingredient of the vaccine 3 of the present invention, is an associate in which the complex protein 3 of the present invention is a monomer, and contains a trimer and / or hexamer of the complex protein 3, or a mixture of the trimer and hexamer. Hereinafter, the substance contained in the associate 3 of the present invention may be collectively referred to as "trimer and / or hexamer 3." In other words, the associate 3 of the present invention is an associate containing a trimer and / or hexamer in which the complex protein 3 of the present invention is a monomer. Furthermore, in consideration of the production process of the complex 3 of the present invention described below, the associate 3 of the present invention can also be defined as an associate formed by associating the complex protein 3 of the present invention in an aqueous liquid. The associate 3 of the present invention is capable of penetrating into cells by itself.

[0034] The trimer is a trimeric protein composed of the same or different complex proteins 3 of the present invention as monomer proteins, and the hexamer is a hexameric protein composed of two molecules of the trimeric protein 3 associated with each other.

[0035] The aggregate 3 of the present invention can be produced by contacting the conjugated protein 3 of the present invention in an aqueous liquid. Depending on the compatibility between the peptide to be supported and the molecular needle, the above-mentioned aggregate may not be formed, or even if an aggregate is formed, it may not be soluble in the aqueous liquid. However, by contacting the target conjugated protein 3 in an aqueous liquid and confirming the results using SDS-PAGE, high-speed atomic force microscopy, gel filtration chromatography, or the like, it is possible to easily determine whether the target conjugated protein forms a trimer or hexamer and can be used as an active ingredient in the vaccine 3 of the present invention. Furthermore, solubility in an aqueous liquid can be easily confirmed by actually conducting a solubilization test.

[0036] [3] Vaccines of the Present Invention (3-1) Vaccine 1 of the Present Invention Vaccine 1 of the present invention is a vaccine containing aggregate 1 of the present invention as an active ingredient (protective antigen), and is a component vaccine for eliciting cellular immunity that contains one or more peptides containing one or more peptides derived from pathogenic microorganisms that match MHC class I as an active ingredient, and is suitable for mucosal, transdermal, subcutaneous, intradermal, or intramuscular administration.

[0037] In other words, vaccine 1 of the present invention is a component vaccine involving "assembling conjugated protein 1 of the present invention as aggregate 1 of the present invention, and using conjugated protein 1 of the present invention or aggregate 1 of the present invention as an active ingredient of vaccine 1 of the present invention to activate cellular immunity."

[0038] In order to further enhance immunogenicity, it is possible to include in the vaccine of the present invention a molecular needle carrying an adjuvant (for example, the B subunit of cholera toxin as the above-mentioned W), but it is preferable that the ability to make an adjuvant-free vaccine by excluding the adjuvant is prioritized as an advantage of vaccine 1 of the present invention.

[0039] The animals to which the vaccine 1 of the present invention can be applied include not only humans but also all animals that can be infected with a specified pathogenic microorganism, such as dogs and cats, but are not limited to these.

[0040] (3-2) Vaccine 2 of the Present Invention Vaccine 2 of the present invention is a vaccine containing aggregate 2 of the present invention as an active ingredient (protective antigen), and is a component vaccine for eliciting humoral immunity that contains one or more peptides, including one or more peptides derived from pathogenic microorganisms that match MHC class II, as active ingredients, and is suitable for mucosal, transdermal, subcutaneous, intradermal, or intramuscular administration.

[0041] Vaccine 2 of the present invention has aggregate 2 of the present invention as an active ingredient (protective antigen), i.e., vaccine 2 of the present invention is a component vaccine relating to "use of conjugate protein 2 of the present invention or aggregate 2 of the present invention to associate conjugate protein 1 of the present invention as aggregate 1 of the present invention and to preferentially activate humoral immunity as the active ingredient of vaccine 2 of the present invention."

[0042] In order to further enhance immunogenicity, it is possible to include in the vaccine of the present invention a molecular needle carrying an adjuvant (for example, the B subunit of cholera toxin as the above-mentioned W), but it is preferable that the ability to make an adjuvant-free vaccine by excluding the adjuvant is prioritized as an advantage of vaccine 2 of the present invention.

[0043] The animals to which the vaccine 2 of the present invention can be applied include not only humans but also all animals that can be infected with a specified pathogenic microorganism, such as dogs and cats, but are not limited to these.

[0044] (3-3) Combination of Vaccines 1 and 2 of the Present Invention Vaccines 1 and 2 of the Present Invention can be used as separate vaccines, or they can be used in combination. That is, aggregates 1 and 2 of the present invention can be mixed to form the active ingredient of the vaccine of the present invention. In this case, the mixing ratio of aggregates 1 and 2 of the present invention can be determined depending on the type of pathogenic microorganism to be protected against and the recipient of the vaccine. As described above, when there is a risk that the administration of a conventional vaccine against a pathogenic microorganism will cause disease enhancement, it is possible to verify whether such disease enhancement is due to cellular immunity or humoral immunity, and select the mixing ratio of vaccines 1 and 2 of the present invention based on the verification results. For example, if verification results indicate that disease enhancement is due to humoral immunity, it is preferable to reduce the mixing ratio of aggregate 2 of the present invention, which preferentially induces humoral immunity, and to use aggregate 1 of the present invention to create a vaccine that primarily induces cellular immunity.

[0045] (3-4) Vaccine 3 of the present invention Vaccine 3 of the present invention is a vaccine that contains aggregate 3 of the present invention as an active ingredient (protective antigen), and is a component vaccine that induces both cellular and humoral immunity and is suitable for mucosal, transdermal, subcutaneous, intradermal, or intramuscular administration, and contains as active ingredients one or more peptides including one or more peptides derived from pathogenic microorganisms that match MHC class I and one or more peptides derived from pathogenic microorganisms that match MHC class II.

[0046] In other words, vaccine 3 of the present invention is a component vaccine involving "use of conjugated protein 3 of the present invention or aggregate 3 of the present invention to activate both cellular immunity and humoral immunity as an active ingredient of vaccine 3 of the present invention by assembling conjugated protein 3 of the present invention as aggregate 3 of the present invention."

[0047] In order to further enhance immunogenicity, it is possible to include in the vaccine of the present invention a molecular needle carrying an adjuvant (for example, the B subunit of cholera toxin as the above-mentioned W), but it is preferable that the ability to make an adjuvant-free vaccine by excluding the adjuvant is prioritized as an advantage of vaccine 3 of the present invention.

[0048] The animals to which the vaccine 3 of the present invention can be applied include not only humans but also all animals that can be infected with a specified pathogenic microorganism, such as dogs and cats, but are not limited to these.

[0049] [4] Method for Producing the Aggregate of the Present Invention The aggregate of the present invention can be produced by contacting three or more molecules of the complex protein of the present invention via an aqueous liquid, causing the complex proteins to associate with each other as monomers to form a mixture of trimers and hexamers, and then selectively separating and collecting the trimers or hexamers as needed.

[0050] The conjugated protein of the present invention can be produced by expressing nucleic acids encoding the conjugated protein by genetic engineering techniques or by synthesizing the conjugated protein by peptide synthesis techniques. When the conjugated proteins are contacted with each other in an aqueous liquid, trimers and hexamers of the conjugated protein spontaneously form, forming a mixture containing the trimers and hexamers. The trimers or hexamers can then be selectively separated and collected, allowing the trimers and hexamers to be produced separately.

[0051] With regard to "aqueous liquids," particularly when the conjugated protein of the present invention is produced by genetic engineering techniques, the conjugated protein is biologically expressed, and the conjugated protein is exposed, for example, by collecting, disrupting, or lysing expressing cells, and then isolating the conjugated protein by a known separation method. In these aqueous liquids, such as water and various buffer solutions, spontaneous association can occur to obtain a mixture containing the trimer and hexamer that are the aggregate of the present invention. Furthermore, the conjugated protein of the present invention, which has been produced, for example, by total chemical synthesis or by separate synthesis of parts followed by bonding by chemical modification, can be suspended in aqueous liquids such as water and various buffer solutions to spontaneously associate, thereby obtaining a mixture containing the trimer and hexamer that are the aggregate of the present invention.

[0052] The method for separating and collecting the trimer or hexamer from the mixture containing the trimer and hexamer is not particularly limited, and examples thereof include known molecular weight fractionation methods, such as gel electrophoresis, affinity chromatography, molecular sieving such as molecular exclusion chromatography, and ion exchange chromatography.

[0053] Therefore, one of the most preferred embodiments of the method for producing the above-mentioned complex is a production method comprising: "culturing a transformant, into which a nucleic acid encoding the complex protein of the present invention has been introduced, in a liquid medium to express the complex protein, and obtaining a mixture containing trimers and hexamers in which the complex protein is a monomer, which are produced by spontaneous assembly; and further selectively separating and collecting the trimers or hexamers from the mixture."

[0054] The aggregate of the present invention thus produced can be used as an active ingredient of the vaccine of the present invention.

[0055] [5] Pathogenic microorganisms to which the present invention can be applied Pathogenic microorganisms for which a peptide compatible with MHC class I or class II can be used as the above-mentioned W include viruses, bacteria, fungi, protozoa, etc.

[0056] The virus may be a DNA virus, an RNA virus, or a retrovirus. Examples include pathogenic viruses for infectious diseases for which vaccines are currently available, such as poliovirus, measles virus, rubella virus, mumps virus, chickenpox virus, yellow fever virus, rotavirus, varicella virus (herpes virus), influenza virus, norovirus, astrovirus, sapovirus, rabies virus, Japanese encephalitis virus, hepatitis virus (hepatitis A, hepatitis B, hepatitis C, etc.), human papillomavirus, HIV, and Ebola virus, as well as pathogenic viruses for infectious diseases for which vaccines are not currently available, such as respiratory syncytial virus (RSV), dengue fever virus, and coronaviruses (including the novel coronavirus). One of the features of the vaccine of the present invention is that it can be effective against infectious diseases for which vaccines are not currently available. For these viruses for which vaccines are currently not available, disease enhancement (cytokine storm) has been reported upon vaccination, which presents a major obstacle to vaccine development. The present invention is applicable to these viruses that carry the risk of disease enhancement upon vaccination.

[0057] Examples of bacteria include pneumococcus, tuberculosis, cholera, shigella, typhoid, anthrax, meningococcus, tetanus, diphtheria, and pertussis.

[0058] Examples of fungi include Trichophyton, Candida, Cryptococcus, and Aspergillus.

[0059] Examples of protozoa include malaria parasites.

[0060] [6] Carrier Peptide (W) (1) Peptide compatible with MHC class I (hereinafter also referred to as class I peptide) Class I peptides usually contain an essential portion consisting of 8-10 amino acid residues. Class I peptides are short peptides characteristic of each target pathogenic microorganism, and bind to the peptide-receptor groove of the MHC class I molecule specific to each class I peptide via several anchor residues, including both ends of the peptide. Even peptides longer than 10 amino acid residues can bind to MHC class I molecules if their C-terminus is capable of binding to MHC class I molecules. In such cases, the peptide is cleaved and shortened by exopeptidases expressed in the endoplasmic reticulum after binding. The peptide-MHC class I complex formed in this way is presented on the cell surface, and this presented peptide-MHC class I complex "is primed by the peptide-MHC class I complex and specifically attacks the complex" (typically CD8 ＋ T cells), and this recognition induces cytotoxic CD8 T cells against cells infected with the target pathogenic microorganism on which the class I peptide is presented. ＋ This signals an attack by T cells, triggering cellular immunity against the target pathogenic microorganism.

[0061] When cellular immunity is induced by class I peptides, cytokines or chemokines released from immunocompetent cells include type I IFN, which is released during the above-mentioned initial stimulation. Other cytokines include IFN-γ, IL-2, IL-12, MIP-1a, MIP-1b, TNF-α, IL-10, perforin, granzyme a, granzyme b, and RANTES, which are released during differentiation into CTLs and during cytotoxicity by CTLs. Detection of these cytokines or chemokines makes it possible to detect the function of cellular immunity via MHC class I.

[0062] Both ends of a class I peptide are anchored to an MHC class I molecule by bonds between the N- and C-terminal atoms of the class I peptide and atoms constituting well-conserved amino acid residues (e.g., clusters of tyrosine residues) at both ends of the peptide-receiving groove of the MHC class I molecule. The primary differences between different MHC class I alleles reside in the coding of specific surfaces of the peptide-receiving groove. Consequently, different MHC class I molecules preferentially bind matching class I peptides due to the different amino acid residues constituting this surface. Class I peptides that bind to specific MHC class I molecules have identical or very similar amino acid residues at two or three positions in their amino acid sequence. The class I peptide fits into the corresponding MHC class I molecule's receiving groove through binding with specific amino acid side chains in the receiving groove that match the side chains of the amino acid residues at these positions in the class I peptide. These specific amino acid residues in the class I peptide are called "anchor residues." Although the location and characteristics of these anchor residues vary, most class I peptides have a hydrophobic (or basic) anchor residue at the C-terminus. Hydrophobic amino acids include aromatic amino acids such as phenylalanine and tyrosine; and hydrophobic amino acids such as valine, leucine, and isoleucine. Basic amino acids include lysine, arginine, and histidine. Furthermore, whether a peptide of an appropriate length containing these anchor residues will bind to an MHC class I molecule is not guaranteed; the binding depends on the properties of amino acid residues at other positions in the peptide; these amino acid residues are called "secondary anchors."

[0063] To select class I peptides, computer programs or databases (e.g., http: / / tools.iedb.org / main / ; http: / / tools.immuneepitope.org / main / datasets / ; T cell epitope prediction by Genetyx software, etc.) with algorithms for selecting the properties of the class I peptides are used to select candidate peptide sequences from the nucleotide or amino acid sequences of the gene encoding the target pathogenic microorganism, and the candidate peptides are then synthesized. The synthetic peptides can then be subjected to a test using cellular immune activation as an indicator to obtain the desired class I peptide. A suitable example of such a test method is the ELISPOT assay. In the ELISPOT assay, infected cells are first fixed in the wells of a multi-well plate, and then the infected cells are transfected with the class I peptide candidate. The transfection method is not limited, and examples include the introduction of a plasmid incorporating the class I peptide candidate via a lipoprotein vehicle, or RNA containing a segment encoding the class I peptide candidate. In the cells transfected with the candidate class I peptide, if the candidate is a class I peptide, it binds to the MHC class I molecule of the infected cell and is presented on the cell surface. Next, the candidate is subjected to immunoprecipitation using leukocytes (typically CD8) from a mammal such as a human whose target disease has been cured. ＋ T cells) are reacted with the leukocytes. These leukocytes attack infected cells that present MHC class I molecules bound to class I peptides on their cell surface, causing the infected cells in the wells where the class I peptides were used to lyse and form plaques. The test peptides that form such plaques are the desired class I peptides. In this way, the desired class I peptides can be determined and obtained.

[0064] As described above, class I peptides can also be selected by detecting cytokines or chemokines characteristic of the cellular immune response mediated by MHC class I. This detection can be performed using a molecular needle carrying the test peptide, as shown in the Examples. This will be described later.

[0065] (2) Peptides Compatible with MHC Class II (hereinafter also referred to as Class II Peptides) Class II peptides are peptides that contain approximately eight or more essential amino acid residues, vary in length, and are characteristic of each target pathogenic microorganism. Each Class II peptide binds to the peptide-receptor groove of a specific MHC Class II molecule via several anchor residues to form a peptide-MHC Class II complex. However, unlike MHC Class I, both ends of a Class II peptide do not bind to an MHC Class II molecule. The number of amino acid residues in a Class II peptide is not limited as long as it contains the essential region for a Class II peptide. The protruding portion is often cleaved by peptidases while fitting into the peptide-receptor groove of an MHC Class II molecule. MHC Class II molecules are present in antigen-presenting cells involved in immune responses, such as dendritic cells, B cells, macrophages, and thymic epithelial cells. The peptide-MHC Class II complex formed in a dendritic cell interacts with naive CD4 ＋ It plays a role in activating T cells. It activates CD4 T cells by absorbing class II peptides presented by peptide-MHC class II complexes formed on B cells. ＋ When T cells recognize the CD4 ＋ T cells (Th2 cells) secrete cytokines to determine the isotype of antibodies to be produced by the B cells, thereby inducing humoral immunity. Meanwhile, activated CD4 ＋ When T cells (Th1 cells) recognize the CD4 ＋T cells activate macrophages and destroy target pathogenic microorganisms present in the endoplasmic reticulum of macrophages, in part via cytokines. In this way, cellular immunity is induced by MHC class II and class II peptides. Cytokines involved in the function of Th1 cells include IFN-γ, IL-12, TNF-α, etc. Cytokines involved in the function of Th2 cells include IFN-γ, IL-12, IL-4, IL-5, IL-6, IL-10, IL-12, etc.

[0066] As will be shown in the Examples below, immunization with class II peptides not only elicits humoral immunity, such as an increase in IgG and IgA antibody titers, but also cellular immunity, such as selective elimination of virus-infected cells, resulting in a synergistic effect that dramatically reduces the number of virus particles in tissues. This indicates that class II peptides not only primarily elicit humoral immunity, but also elicit cellular immunity through the intercellular network resulting from the secretion of physiologically active substances.

[0067] Unlike class I peptides, class II peptides do not contain amino acid residues at either end of the peptide that are important for binding to MHC class II molecules, and as mentioned above, these regions do not bind to MHC class II molecules. Instead, class II peptides are extended and fit into the peptide-binding groove of MHC class II molecules. The peptide side chains fit into a pocket formed by highly polymorphic amino acid residues, and the peptide backbone binds to the side chains of amino acid residues that are well conserved in the peptide-binding groove of all MHC class II molecules, thereby anchoring the class II peptide in the peptide-binding groove. Analysis has shown that the side chains of amino acids defined as positions 1, 4, 6, and 9 of class II peptides fit into these pockets, with position 1 tending to be hydrophobic amino acid residues (tyrosine, leucine, phenylalanine, and isoleucine), position 4 tending to be negatively charged amino acid residues (aspartic acid and glutamic acid), position 6 tending to be basic amino acid residues (lysine, asparagine, histidine, and glutamine), and position 9 tending to be hydrophobic amino acid residues (tyrosine, leucine, phenylalanine, and isoleucine). However, these are merely trends, and exceptions are possible. Because the pockets of MHC class II molecules can accommodate a wide variety of amino acids, it is difficult to determine the anchor residues of class II peptides and predict which peptides will bind to specific MHC class II molecules. However, by comparing the amino acid sequences of class II peptides that are already known to bind, it is possible to analyze the patterns of amino acid residues common to class II peptides that bind to MHC class II molecules derived from different alleles, and further to predict the sequence motifs of amino acid residues of peptides that bind to the pocket portion of the peptide-receiving groove of MHC class II molecules.

[0068] To select class II peptides, commercially available computer programs or databases (e.g., http: / / tools.iedb.org / main / ; http: / / tools.immuneepitope.org / main / datasets / ; http: / / www.cbs.dtu.dk / services / NetMHCIIpan / logos.php; Genetyx software's T cell epitope prediction, etc.) with algorithms for selecting the properties of the above-mentioned class II peptides are used to select candidate peptide sequences from the nucleotide or amino acid sequences of the gene encoding the target pathogenic microorganism, and the candidate peptides are then synthesized. The synthetic peptides can then be subjected to a test using humoral immune activation as an indicator to obtain the desired class II peptide. Examples of such a test method include the ELISA method (indirect method). Specifically, for example, a class II peptide candidate is fixed in the well of a multi-hole plate, and then serum (polyclonal antibody) from a mammal such as a human that has been cured of the target disease is contacted thereon, and then a labeled secondary antibody against the antibody is contacted thereon, and evaluation is performed based on the label, making it possible to select whether the class II peptide candidate is the desired class II peptide.

[0069] As described above, class II peptides can also be selected by detecting cytokines characteristic of MHC class II activity. This detection can be performed using a molecular needle carrying the test peptide, as shown in the Examples. This will be described later.

[0070] (3) Linked Peptides of Class I and Class II Peptides Class I peptides and Class II peptides can be linked together, either separately or together, to form a peptide (hereinafter also referred to as a linked peptide) that can be used as the supported peptide (W). By selecting the type and number of constituent peptides in the linked peptide used as the supported peptide, it is possible to adjust the type and strength of immunity induced by a vaccine containing a molecular needle carrying such a linked peptide as an active ingredient. Increasing the number of class I peptides can increase the strength of cellular immunity, and increasing the number of class II peptides can increase the strength of humoral immunity.

[0071] A vaccine of the present invention, which contains as an active ingredient a molecular needle carrying a connecting peptide whose only component is a class I peptide, is a form of vaccine 1 of the present invention. The individual class I peptides constituting the connecting peptide may be the same or different. It is preferable that the individual class I peptides are linked to each other via a linker peptide. The number of amino acid residues constituting such a linker is not limited, but is usually 3 to 10. Furthermore, the number of class I peptides constituting the sequence peptide is not particularly limited, but is usually preferably 2 to 15, and more preferably 2 to 5.

[0072] A vaccine of the present invention, which contains as an active ingredient a molecular needle carrying a connecting peptide whose only component is a class II peptide, is a form of vaccine 2 of the present invention. The individual class II peptides constituting the connecting peptide may be the same or different. It is preferable that the individual class II peptides are linked to each other via a linker peptide. The number of amino acid residues constituting such a linker is not limited, but is usually 3 to 10. Furthermore, the number of class II peptides constituting the sequence peptide is not particularly limited, but is usually preferably 2 to 15, and more preferably 2 to 5.

[0073] A vaccine of the present invention, which contains as an active ingredient a molecular needle carrying a connecting peptide composed of both a class I peptide and a class II peptide, is a form of vaccine 3 of the present invention. The individual class I peptides and class II peptides constituting the connecting peptide may be the same or different. The individual class I peptides and class II peptides are preferably linked to each other via a linker peptide. The number of amino acid residues constituting such a linker is not particularly limited, but is typically 3 to 10. The number of class I peptides and class II peptides constituting the sequence peptide is also not particularly limited, but is typically 2 to 15, and more preferably 2 to 5.

[0074] (4) Peptides with the Functions of Both Class I and Class II Peptides In living organisms, cellular and humoral immunity function in a balanced manner during microbial infection. In particular, when a peptide contains more than eight amino acids, which could be a Class II peptide, the peptide may contain both Class I and Class II peptide motifs overlapping or as separate motifs. As mentioned above, when a peptide functions as a Class I peptide, it is thought that the excess portion of the peptide other than the Class I peptide itself is digested by exopeptidases in the endoplasmic reticulum.

[0075] A peptide that functions as both a class I peptide and a class II peptide has at least eight amino acids, which is the minimum number of amino acids required for a class II peptide.

[0076] [7] Information Acquisition Method of the Present Invention As described above, when determining an immunogen for a vaccine, it is extremely important in the development of a new vaccine to identify a peptide or protein derived from a pathogenic microorganism that is unlikely to cause a cytokine storm, etc. Furthermore, as described above, the importance of acquiring information on whether an immunogen belongs to MHC class I or class II has already been mentioned.

[0077] The present inventors have conceived the idea of ​​solving the problems of suppressing the cytokine storm and determining the MHC class by immunizing animals with molecular needles carrying test peptides or proteins and determining changes in the secretion of physiologically active substances such as cytokines after the immunization.

[0078] That is, the present invention includes the following information acquisition method (hereinafter also referred to as the information acquisition method of the present invention).

[0079] The information acquisition method of the present invention comprises the steps of: "[A] obtaining an amino acid sequence of the following formula (1-2): W ２ -L １ -X ｎ -Y (1-2) [wherein, W ２ represents the amino acid sequence of a peptide or protein derived from a pathogenic microorganism that is the test immunogen, and L １ represents a first linker sequence having 0-100 amino acids, X represents the amino acid sequence of SEQ ID NO: 1, Y represents the amino acid sequence of the cell introduction region, and n, which is the number of repetitions of X, is an integer of 1-10.], and the amino acid sequence of the cell introduction region Y is represented by the following formula (2): Y １ -L ２ -Y ２ -Y ３ (2) [wherein, Y １ represents any one amino acid sequence selected from the group consisting of SEQ ID NOs: 2-5, and Y ２ represents any one amino acid sequence selected from the group consisting of SEQ ID NOs: 6-9, and L ２ represents a second linker sequence of 0-30 amino acids, and Y ３ indicates the amino acid sequence for modification, and Y ２ or Y ３ In some cases, X may not be present. ｎ , Y １ , or Y ２a trimer or hexamer of a conjugated protein, which is permitted to contain a modified amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by [A] above; a test animal is immunized with the trimer or hexamer represented by [A] above, immunocompetent cells of the immunized test animal are separated from the test animal outside the body, one or more physiologically active substances in the separated immunocompetent cells are quantified, the separated immunocompetent cells are then infected with a target pathogenic microorganism, the physiologically active substances in the immunocompetent cells after the infection are quantified, and the amount of each physiologically active substance before and after the infection, obtained from the first two quantitative values, is used as an index to determine the amount of the test immunogen W ２ "A method for obtaining information regarding the secretion of physiologically active substances after immunization in a mammal."

[0080] The information acquisition method of the present invention includes, for example, "immunizing a test animal with the trimer or hexamer represented by [A] above, isolating immunocompetent cells from the immunized test animal outside the body, quantifying one or more physiologically active substances in the isolated immunocompetent cells, infecting the isolated immunocompetent cells with a target pathogenic microorganism, quantifying the physiologically active substances in the infected immunocompetent cells, and using the change in the amount of each physiologically active substance before and after the infection, which is obtained from the first two quantitative values, as an index, to obtain a test immunogen W. ２ The present invention can also be expressed as "a detection method for detecting the secretion state of a physiologically active substance after immunization in a mammal."

[0081] W ２ is "an amino acid sequence of a peptide or protein derived from a pathogenic microorganism that is a test immunogen." １ , X, Y, n, Y １ , L ２ , Y ２ , and Y ３ " is the same as "

[0082] Immunocompetent cells are obtained by collecting them from biological tissues where they are present, such as the spleen, bone marrow, thymus, and lymph nodes, or from biological fluids such as blood and lymph. The subject animal that is the target of the information acquisition method of the present invention is not particularly limited, but preferred examples include mammals such as rats, mice, hamsters, monkeys, dogs, and cats. Humans can also be used as the target, but the immunocompetent cells must be collected from biological fluids (blood, lymph, spinal fluid, etc.) isolated from a living body or biological tissue isolated from a living body during a biopsy or a life-saving or surgical procedure.

[0083] Immunocompetent cells include, but are not limited to, T cells, B cells, macrophages, mast cells, eosinophils, neutrophils, basophils, dendritic cells, and mixtures of these cells.

[0084] The physiologically active substance from which information is obtained is not particularly limited as long as it is technically detectable, but it is preferable that it includes cytokines or chemokines that are secreted as the immune system of the living body progresses.

[0085] As shown in the Examples below, information on the secretion of physiologically active substances can be obtained by ２ In one preferred embodiment, the information is information about whether the antibody belongs to MHC class I or MHC class II.

[0086] As described above, signals from a test immunogen belonging to MHC class I include activation signals such as IFN-γ, IL-2, IL-12, MIP-1a, MIP-1b, TNF-α, IL-10, perforin, granzyme a, granzyme b, and RANTES. Furthermore, signals from a test immunogen belonging to MHC class II include activation signals from IFN-γ, IL-12, and TNF-α as cytokines involved in the function of Th1 cells; and activation signals from IFN-γ, IL-12, IL-4, IL-5, IL-6, IL-10, and IL-12 as cytokines involved in the function of Th2 cells. The activation signal is typically a change in the amount of secretion of a physiologically active substance due to infection with a target pathogenic microorganism, and this change is typically an increase. There are no particular limitations on the threshold at which this increase is considered a positive signal. For example, as in the Examples, a suitable threshold for detecting activation of secretion of a physiologically active substance without omission and without noise is, for example, an activation signal when the secretion amount after infection exceeds twice that before infection, but the threshold can be freely determined depending on the purpose of information acquisition, the type and number of physiologically active substances to be detected, etc. Furthermore, particularly when there are many types and numbers of physiologically active substances to be detected, for example, five or more types, exceeding the threshold for all of the set physiologically active substances is not a prerequisite for evaluation of, for example, MHCI type I or II, but if approximately 80%, for example, four out of five types, exceed the threshold, it can be a prerequisite for a definitive evaluation of the MHC type.

[0087] As in other aspects of the present invention, pathogenic microorganisms that are targets of the information acquisition method of the present invention are exemplified by viruses, bacteria, fungi, protozoa, etc., and among these, viruses are one of the preferred aspects.

[0088] Information obtained by the information obtaining method of the present invention regarding the secretion characteristics of physiologically active substances during infection with a target microorganism of a test immunogen is important information when selecting a vaccine immunogen, for example, for selecting a more effective immunogen by suppressing cytokine storm or selecting MHC I or II. Evaluation of MHC I or II is also useful as a confirmation evaluation of prior analysis using computer software or prior selection based on known information. The information obtaining method of the present invention is useful not only when the type of active ingredient of the vaccine being developed is a peptide or protein, but also when it is a nucleic acid.

[0089] According to the present invention, (1) a molecular needle containing the immunogen "one or more peptides containing one or more peptides derived from a pathogenic microorganism compatible with MHC class I," "one or more peptides containing one or more peptides derived from a pathogenic microorganism compatible with MHC class II," or "one or more peptides containing a peptide derived from a pathogenic microorganism compatible with MHC class I and one or more peptides derived from a pathogenic microorganism compatible with MHC class II" can be used as a basic unit of the active ingredient (protective antigen) of a component vaccine that efficiently introduces the immunogen "one or more peptides containing one or more peptides derived from a pathogenic microorganism compatible with MHC class I" into cells of a target tissue by mucosal, transdermal, subcutaneous, intradermal, or intramuscular administration. (1) a composite protein (monomer) carrying a peptide compatible with MHC class I and / or MHC class II; (2) an aggregate of the composite protein that can be used as the active ingredient (protective antigen); (3) an MHC class I-agonistic component vaccine (vaccine 1 of the present invention) that selectively induces cellular immunity, an MHC class II-agonistic component vaccine (vaccine 2 of the present invention) that preferentially induces humoral immunity, or an MHC class I and II-agonistic component vaccine (vaccine 3 of the present invention) that induces both cellular and humoral immunity, each of which contains the aggregate as an active ingredient; and also associated gene expression vectors and transformants. Furthermore, (4) a method for obtaining information on the secretion of physiologically active substances, which is extremely useful for evaluating the secretion characteristics of physiologically active substances such as cytokines during infection with a target pathogenic microorganism and for evaluating MHC types, is provided.

[0090] 1 is a diagram showing the process of constructing trimers and hexamers, which are complexes of the present invention, based on the complex protein of the present invention. FIG. 1 shows the results of MALDI-TOF mass spectrometry for PN-sRL-VPg, where (a) shows the signal for the monomer, (b) shows the signal for the trimer, and (c) shows the signal for the hexamer (trimer-dimer), with each section showing a schematic diagram illustrating the complex protein (monomer) of the present invention. FIG. 1 is a diagram showing the gene structure of respiratory syncytial virus. 1 is a schematic diagram of the plasmid construct "pET29b(+) / Lpep-PN," prepared by replacing the gene fragment encoding the test peptide of Example 1 with Vpg by the InFusion cloning method.

[0034] FIG. 1 is a diagram showing the IgA induction effect of nasal inoculation of a component vaccine used in Example 1, which contains as an active ingredient the complex protein assembly of the present invention, carrying a peptide containing a test peptide in the L protein, one of the nonstructural proteins of RS virus, three weeks after inoculation.

[0035] FIG. 1 is a diagram showing the IgG induction effect of nasal inoculation of a component vaccine used in Example 1, which contains as an active ingredient the complex protein assembly of the present invention, carrying a test peptide in the L protein, one of the nonstructural proteins of RS virus, three weeks after inoculation.

[0036] FIG. 1 is a diagram showing the suppression effect of RS virus count in the lungs of nasal inoculation of a component vaccine used in Example 1, which contains as an active ingredient the complex protein assembly of the present invention, carrying a test peptide in the L protein, one of the nonstructural proteins of RS virus. 1 is a diagram examining the change in body weight over time after infection of test hamsters with the novel coronavirus in Example 2. FIG. 2 is a diagram examining the amount of virus in the lung tissue of test hamsters with the novel coronavirus in Example 2.

[0091] (1) The conjugated protein of the present invention. Formula (1) is an amino acid sequence formula representing the conjugated protein of the present invention: W-L １ -X ｎ -Y (1) [wherein W represents one or more amino acid sequences of peptides containing one or more peptides derived from pathogenic microorganisms compatible with MHC class I and / or one or more peptides derived from pathogenic microorganisms compatible with MHC class II, which are immunogens; １ represents a first linker sequence having 0-100 amino acids, X represents the amino acid sequence of SEQ ID NO: 1, Y represents the amino acid sequence of the cell introduction domain, and n is an integer of 1-3. As described above, the immunogen W is one or more amino acid sequences of peptides containing one or more peptides derived from pathogenic microorganisms compatible with MHC class I and / or one or more peptides derived from pathogenic microorganisms compatible with MHC class II. With regard to this immunogen, the "two or more (plural)" in "one or more peptides derived from pathogenic microorganisms" refers, for example, to a case where a single peptide contains multiple class I peptides and / or class II peptides in a linked form or the like. Furthermore, the term "comprises" includes, for example, a case where, in addition to the original class I peptide or class II peptide, an amino acid sequence for modification is artificially linked, or a linker amino acid sequence is linked to form W.

[0092] Regarding class I peptides and class II peptides, it is possible to link only class I or only class II, or it is also possible to link both class I and class II peptides. The number of amino acid residues in the linker peptide used for linking is preferably 3-10, more preferably 4-6. The number of class I peptides and / or class II peptides constituting the linking peptide is preferably 2-15. Specific examples of linker peptides include, but are not limited to, "GGGG" (SEQ ID NO: 58), "GGGGS" (SEQ ID NO: 15), "PAPAP" (SEQ ID NO: 16), and "SNSSSVPGG" (SEQ ID NO: 14) (single-letter amino acid code).

[0093] For example, the same class I peptide or class II peptide (W1) can be linked with a linker peptide, such as "W1 + GGGGS + W1 + GGGGS + W1," to form the above W. Alternatively, different class I peptides and / or class II peptides can be linked with a linker peptide, such as "W1 + GGGGS + W2 + GGGGS + W3."

[0094] L represents the first linker sequence １ is necessary to maintain an appropriate distance between the immunogen W and the molecular needle portion Y to suppress steric hindrance, and the number of amino acid residues is 0 to 100, preferably 4 to 40, as described above. The specific sequence is not limited, but for example, (GGGGS) ｍ , (PAPAP) ｍ ,(SNSSSVPGG) ｍ [m is the number of repetitions, and is preferably an integer of 1 to 10, and particularly preferably 1 to 3], etc. However, these are merely examples.

[0095] X is the amino acid sequence X consisting of the amino acids of SEQ ID NO: 1 ｎ The repeating unit is a sequence of n (integer) repeating units of X in the above. The repeating form is a series repeat, for example, X ２ If the repeat sequence X is "XX" ("-" represents a peptide bond), ｎ In the above, the above-mentioned amino acid sequence modifications are allowed. Here, n is an integer of 1 to 3 as described above, and is preferably 1, but may also be 2 or 3. ｎ When n is 2 or 3, the main purpose is to stably maintain the distance between the molecular needles Y at an appropriate distance depending on the size and characteristics of the immunogen W.

[0096] The cell introduction region Y corresponds to the basic structure of a molecular needle and is based on the needle part (cell introduction part) of the tail of a bacteriophage, and is represented by the formula (2): Y １ -L ２ -Y ２ -Y ３ (2) [wherein, Y １represents an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-5, and Y ２ represents an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-9, and L ２ represents a second linker sequence of 0-30 amino acids, and Y ３ represents the amino acid sequence for a given modification, and Y ２ or Y ３ may not be present.]

[0097] Y in formula (2) １ Of these, the first 32 amino acids (32 Leu) on the N-terminal side are the amino acid sequence of the triple helix β-sheet structure of bacteriophage T4. At least the N-terminal amino acid valine (1 Val) may be replaced with leucine (1 Leu). The remaining C-terminal side is the amino acid sequence of the C-terminal part of the needle protein of the bacteriophage. １ Examples of amino acid sequences that can be used at the C-terminus of Y include the amino acid sequence of gp5 of bacteriophage T4, the amino acid sequence of gpV of bacteriophage P2, the amino acid sequence of gp45 of bacteriophage Mu, and the amino acid sequence of gp138 of bacteriophage φ92. More specifically, a Y having the amino acid sequence of gp5 of bacteriophage T4 at the C-terminus thereof can be used. １ The amino acid sequence of SEQ ID NO: 2 is a Y having the amino acid sequence of gpV of bacteriophage P2 on the C-terminal side. １ The amino acid sequence of SEQ ID NO: 3 is a Y having the amino acid sequence of gp45 of bacteriophage Mu on the C-terminal side. １ The amino acid sequence of SEQ ID NO: 4 is a Y having the amino acid sequence of gp138 of bacteriophage φ92 on the C-terminal side. １ The amino acid sequence of SEQ ID NO: 5 is an example of this Y. １ Nucleic acid sequences encoding the amino acid sequences of can be selected in accordance with known relationships between amino acids and nucleic acid bases.

[0098] In formula (2), Y ２is the amino acid sequence of a region called the foldon of bacteriophage T4, or the amino acid sequence of a region called the tip of bacteriophage P2, bacteriophage Mu, or bacteriophage φ92. The foldon or tip is a region that constitutes the tip of the molecular needle structure called fibritin of the bacteriophage. ２ Although it is not essential that Y be present, the incorporation efficiency of the molecular needle into the cell membrane can be improved by having the amino acid sequence of the foldon or tip. ２ The amino acid sequence of bacteriophage T4 foldon is shown in SEQ ID NO: 6. A nucleic acid sequence encoding this amino acid sequence can be selected in accordance with known relationships between amino acids and nucleic acid bases.

[0099] The amino acid sequence of the tip of bacteriophage P2 is shown in SEQ ID NO: 7. A nucleic acid sequence encoding this amino acid sequence can be selected in accordance with the known relationships between amino acids and nucleic acid bases. The amino acid sequence of the tip of bacteriophage Mu is shown in SEQ ID NO: 8. A nucleic acid sequence encoding this amino acid sequence can be selected in accordance with the known relationships between amino acids and nucleic acid bases. The amino acid sequence of the tip of bacteriophage φ92 is shown in SEQ ID NO: 9. A nucleic acid sequence encoding this amino acid sequence can be selected in accordance with the known relationships between amino acids and nucleic acid bases.

[0100] L ２ is the Y １ and Y ２ The linker L is a second linker interposed between ２ The number of amino acids in the linker is 0 to 30, preferably 0 to 5. The number of amino acids in the linker is 0 when the second linker L ２ indicates that it does not exist.

[0101] Y ３is an amino acid sequence for modification, which can be selectively added to Y. The amino acid sequence for modification is added for the purpose of protein purification or protection, and examples thereof include tag peptides such as histidine tag, GST tag, and FLAG tag. ３ The inclusion of a histidine tag is preferable in the protein purification process and from the viewpoint of the kinetics when the trimer or hexamer of the conjugated protein is introduced into target cells as an active ingredient of a vaccine. ３ A linker sequence can be added to Y as appropriate, and such a linker sequence itself can also be used as Y ３ can be a component of the amino acid sequence of

[0102] The conjugated protein of the present invention can be produced by known methods, specifically, genetic engineering techniques or chemical synthesis methods. It is also possible to produce the entire conjugated protein of the present invention together, or to produce it in parts and then bond the parts to each other later by chemical modification. １ or L ２ The binding of proteins to each other via a linker having a succinimide group or a maleimide group can be achieved by binding lysine residues or cysteine ​​residues in the two proteins to each other via a linker having a succinimide group or a maleimide group.

[0103] In genetic engineering techniques, nucleic acids encoding all or part of the conjugated protein of the present invention to be produced can be expressed in host cells such as Escherichia coli, yeast, insect cells, or animal cells as transformants, or in cell-free expression systems such as Escherichia coli extract, rabbit reticulocyte extract, or wheat germ extract. Expression vectors incorporating these nucleic acids can be selected according to the expression system, and examples include pET for expression in Escherichia coli, pAUR for expression in yeast, pIEx-1 for expression in insect cells, pBApo-CMV for expression in animal cells, and pF3A for expression in wheat germ extract.

[0104] As the chemical synthesis method, known methods for chemically synthesizing peptides can be used. That is, all or part of the conjugated protein of the present invention can be produced using liquid-phase peptide synthesis or solid-phase peptide synthesis, which are established as conventional methods. Furthermore, solid-phase peptide synthesis, which is generally recognized as a suitable chemical synthesis method, can also be performed using the Boc solid-phase method or the Fmoc solid-phase method, and as described above, a ligation method can also be used if necessary. Furthermore, individual amino acids can be produced by known methods, and commercially available products can also be used.

[0105] (2) Aggregates of the Present Invention Figure 1 shows the process of constructing trimers and hexamers, which are aggregates of the present invention, based on the conjugated protein of the present invention. In Figure 1, 10 is the protein of the present invention as a monomer, 20 is the trimer of the present invention, and 30 is the hexamer of the present invention.

[0106] The conjugated protein 10 of the present invention is "X of formula (2) ｎ and Y １ "Basic part 131 corresponding to "Y of formula (2) ２ The "molecular needle region 13 corresponding to Y in formula (1)" and the "immunogen 11 corresponding to W in formula (1)" are linked to "L in formula (1) １ The linker other than the linker 12 is bonded to the Y ３ The modified sequence corresponding to the following is not shown in the figure. The conjugated protein 10 of the present invention itself does not substantially have the ability to pass through the cell membrane of cells in the target tissue.

[0107] The trimer 30 is a trimer formed by spontaneously associating three monomers of the above-mentioned conjugated protein 10. In the trimer 30, three of the above-mentioned molecular needle regions 13 come together and associate with each other at their C-termini, forming a trimer parallel β-sheet structure and a needle-like structure called a helical structure (triple helix β-sheet structure) by the β-sheet structure itself, forming a molecular needle 13 x 3. The molecular needle 13 x 3 is composed of a basic portion 131 x 3 and a foldon assembly 132 x 3. In this way, a "molecular needle" capable of passing through the cell membrane of cells in the target tissue is formed by trimerization and self-organization, and three linkers (12) derived from each monomer are connected. １ , 12 ２ , 12 ３ ) and three immunogens (11) linked to each of these linkers. １ , 11 ２ , 11 ３ ) exists outside this molecular needle 13×3.

[0108] The hexamer 60 is formed by two units of the trimer 30, each of which is connected to the basic part of the molecular needle ((13 × 3) １ and (13 x 3) ２ ) at the N-terminus, and the hexamer 60 also has the function of passing through the cell membrane of the cells of the target tissue. １ , 12 ２ , 12 ３ , and 12 ５ , 12 ６ :12 ４ (not shown) and six immunogens (11) each bound to these linkers. １ , 11 ２ , 11 ３ , and 11 ５ , 11 ６ :11 ４ (not shown) are two molecular needles (13 × 3) １ and (13 x 3) ２ exists outside of

[0109] The trimerization of the conjugated protein 10 of the present invention to the trimer 30 and the macroscopic dimerization of the trimer 30 to the hexamer 60 proceed spontaneously in an aqueous liquid, resulting in a stable existence as a trimer or hexamer. The stability of this trimer or hexamer is extremely high, for example, even in an aqueous liquid environment at a temperature of 100°C, an aqueous liquid environment at a pH of 2-11, and an aqueous liquid environment containing 50-70% by volume of an organic solvent, and furthermore, it is highly safe. Even when isolated from an aqueous liquid and dried, the trimer or hexamer exhibits excellent stability and cell membrane permeability.

[0110] As described above, the transition from the complex protein of the present invention to the aggregate proceeds spontaneously, and usually the majority of the protein forms a hexamer, which is the final form, but a portion remains as a trimer.

[0111] (3) Vaccines of the Present Invention Due to the excellent cell permeability and immunogenicity of the aggregate of the present invention, which is its active ingredient, the vaccine of the present invention efficiently delivers, via subcutaneous, intradermal, transdermal, mucosal, or intramuscular administration, one or more immunogens, i.e., one or more peptides containing one or more peptides derived from pathogenic microorganisms compatible with MHC class I (Vaccine 1 of the Present Invention), one or more peptides containing one or more peptides derived from pathogenic microorganisms compatible with MHC class II (Vaccine 2 of the Present Invention), or one or more peptides containing a peptide derived from pathogenic microorganisms compatible with MHC class I and one or more peptides derived from pathogenic microorganisms compatible with MHC class II (Vaccine 3 of the Present Invention), to cells of target tissues. Vaccine 1 of the present invention is capable of selectively eliciting cellular immunity, while vaccine 2 of the present invention is capable of preferentially eliciting humoral immunity. Furthermore, vaccine 3 of the present invention is capable of eliciting both cellular immunity and humoral immunity while adjusting the balance between the two. Furthermore, the efficacy and safety of component vaccines against pathogenic microorganisms such as viruses and bacteria can be improved by subcutaneous, intradermal, transdermal, mucosal, or intramuscular administration. This is manifested by the fact that they can be used as adjuvant-free vaccines. The target mucosal tissue for mucosal administration can be freely determined depending on the properties of the target pathogenic microorganism, particularly the affected site of the virus, and includes, but is not limited to, the nasal mucosa, throat mucosa, oral mucosa, bronchial mucosa, gastrointestinal mucosa, vaginal mucosa, etc. In the case of viruses that cause respiratory tract inflammation (cold), such as respiratory syncytial virus, coronaviruses (including betacoronaviruses such as the novel coronavirus), and influenza viruses, the nasal mucosa, throat mucosa, oral mucosa, bronchial mucosa, sublingual mucosa, pulmonary mucosa, etc. are preferred.

[0112] The vaccine of the present invention is provided as a pharmaceutical composition for subcutaneous, intradermal, transdermal, mucosal, or intramuscular administration, which contains the above-mentioned aggregate of the present invention as an active ingredient (protective antigen). Even in the case of direct administration of the aggregate of the present invention, the aggregate is suspended in a buffer solution or the like just before use to form a liquid for subcutaneous, intradermal, transdermal, mucosal, or intramuscular administration, and therefore the form of the aggregate itself is also included in the pharmaceutical composition. Suitable dosage forms for the above-mentioned mucosal administration include aerosols (aerosols, sprays, etc.), capsules, and topical applications.

[0113] The vaccine of the present invention may contain different types of the aggregate of the present invention together as active ingredients.

[0114] The vaccines of the present invention are prepared in the form of a pharmaceutical composition by blending the aggregate of the present invention, which is an essential active ingredient (protective antigen), and, if necessary, a molecular needle carrying other constituent peptides of the target pathogenic microorganism, and, if necessary, an adjuvant or pharmaceutical carrier. However, adjuvant-free formulations are also possible. The pharmaceutical carrier can be selected depending on the form of use, and excipients or diluents such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants can be used. The composition is generally in the form of a liquid, but can also be in the form of a desiccant, powder, granules, or the like for dilution with liquid before use. Furthermore, as described above, the above-mentioned pharmaceutical compositions can also be used as vaccines of the present invention in the form of a vaccine containing the aggregate of the present invention as an active ingredient.

[0115] The amount of the aggregate of the present invention in the vaccine of the present invention (the total amount of the aggregate of the present invention when it is further contained, and the total amount of aggregates of molecular needles carrying constituent proteins or peptides of other pathogenic microorganisms when it is further contained) is selected appropriately and is not fixed, but it is usually preferable to use the aggregate of the present invention as a liquid formulation containing 1-10% by mass at the time of administration. An appropriate administration (inoculation) amount is approximately 0.01 μg-10 mg per adult, and one or more administrations (inoculations) can be performed by appropriately combining a primary inoculation and a booster inoculation as needed.

[0116] When the aggregates of the present invention are used together, the blending ratio of the two aggregates can be selected depending on the type of target pathogenic microorganism and the purpose. For example, if the target pathogenic microorganism tends to be associated with disease enhancement, but it is desired to induce humoral immunity as well as cellular immunity, an effective amount of aggregate 1 of the present invention, which induces cellular immunity, can be used as the main active ingredient, and a small amount of aggregate 2 can be used as a supplementary active ingredient. The specific blending ratio can be determined through individual and specific considerations.

[0117] Examples of the present invention will be disclosed below.

[0118] The purpose of this example is to demonstrate the usefulness of aggregate 2 of the present invention as an active ingredient in a component vaccine targeting a virus. Respiratory syncytial virus (RSV), also known as orthopneumovirus, was selected as the target for this demonstration, taking into account the current difficulty of vaccine production and its usefulness.

[0119] Respiratory syncytial virus (RSV) is widely distributed throughout the world and causes lifelong overt infection in people of all ages. However, it is a particularly important pathogen in infants, causing the most severe symptoms in the first few weeks to months of life despite the presence of maternal antibodies. Low birth weight infants, those with underlying cardiopulmonary diseases, or immunodeficiencies are at higher risk of developing severe symptoms, and the clinical and public health impact is significant.

[0120] Currently, there is no approved RSV vaccine. Clinical trials using a formalin-inactivated vaccine were conducted in the past, but the vaccinated group experienced worse symptoms than the control group, resulting in failure. The only medication available for preventing RSV infection is the humanized monoclonal antibody palivizumab, and the arrival of an effective vaccine is eagerly awaited (above, quoted from the website of the National Institute of Infectious Diseases).

[0121] [Reference Example] Preparation of a vector containing nucleic acid encoding a conjugated protein, which serves as a vector template for producing the complex used in the Examples

[0122] (a) Prerequisites for Template Vector Preparation Genetic engineering techniques were used to prepare vectors incorporating nucleic acids encoding a complex protein whose immunogen is "viral protein genome-linked (Vpg)," a nonstructural protein of the LM14-2 strain of human norovirus GII.4. Vpg is a nonstructural protein contained in open reading frame 1 (ORF1) within the norovirus genome. ORF1 encodes a series of norovirus nonstructural proteins, including the N-terminal protein, NTPase (p48), p22 (3A-like), Vpg, protease, and RNA-dependent RNA polymerase (RdRp). After translation of the entire ORF1, the protease cleaves each nonstructural protein into its mature product. Of these mature products, VPg has been demonstrated to play an essential role in norovirus genome replication through translation from genomic and subgenomic RNAs and functions as a cap substitute during ribosome recruitment. The amino acid sequence of Vpg of the LM14-2 strain, the immunogen used in this example, is as shown in SEQ ID NO: 10 (wherein the N-terminal Met is derived from the start codon ATG). The nucleic acid sequence encoding this can be selected according to the known relationship between amino acids and nucleic acid bases.

[0123] All reagents used in this preliminary step were purchased from commercial suppliers and used without further purification. The HNV-VPg gene fragment was contained in the cDNA portion (7639 bases: SEQ ID NO: 12) of the human norovirus LM14-2 strain, which had been incorporated into the plasmid pHuNoV-LM14-2F (bases 1-12774: SEQ ID NO: 11), provided by Dr. Katayama of the Institute of Viral Infection, Kitasato University. VPg is a 399-base sequence (SEQ ID NO: 13) corresponding to bases 2630 to 3028 of the cDNA portion (7639 bases) of the LM14-2 strain. A start codon, ATG, was added to the 5' end of this sequence for expression.

[0124] UV-vis spectra were measured using a SHIMADZU UV-2400PC UV-vis spectrometer. MALDI-TOF mass spectra were measured using a Bruker Ultraflex Xtreme. For MALDI-TOF-MS measurements, samples were mixed with an equal volume of 70% (v / v) acetonitrile / water solution containing 0.03% (w / v) sinapinic acid and 0.1% (v / v) trifluoroacetic acid. Gel permeation chromatography (GPC) was performed using an HPLC system and column (Asahipack GF-510HQ, Shodex, Tokyo, Japan).

[0125] (b) Construction and expression of the "PN-Vpg" expression plasmid (b)-1: General remarks Here, "PN-Vpg" refers to the vector represented by the formula (3): W １ -L １ -X ｎ -Y (3) and formula (4) representing the cell introduction region Y of formula (3): Y １ -L ２ -Y ２ -Y ３ (4) [L １ , X ｎ , Y, Y １ , L ２ , Y ２ , and Y ３ is the same as in equations (1) and (2), and W １ is an immunogen.] where W is the immunogen １ is "LM14-2 strain-Vpg" represented by the amino acid sequence of SEQ ID NO: 10; the first linker L １ is the amino acid sequence of SEQ ID NO: 14 (SNSSSVPGG), 15 (GGGGS), or 16 (PAPAP); ｎ The repeating unit of is the amino acid sequence of SEQ ID NO: 1, and the number of repeats n is 1; the main body portion Y １ is the amino acid sequence of SEQ ID NO: 2; and the second linker L ２ is "SVE"; Foldon Y ２ is the amino acid sequence of SEQ ID NO: 6; ３ The amino acid sequence of SEQ ID NO: 17 (VEHHHHHH).

[0126] The PN-VPg plasmid was constructed using a flexible linker (FL:SNSSSVPGG (SEQ ID NO: 14)) as a template, and two types of linkers were constructed based on this: a short flexible linker (sFL:GGGGGS (SEQ ID NO: 15)) and a short rigid linker (sRL:PAPAP (SEQ ID NO: 16)). These were expressed, and the contents of the spontaneously formed aggregates were analyzed, confirming that they contained trimers and hexamers. Based on these findings, we then produced and tested a respiratory syncytial virus component vaccine, which was our immediate goal. We demonstrated that aggregate 3 of the present invention, which uses peptides that function as both RS virus MHC class I and class II peptides, is extremely useful as a component vaccine.

[0127] (b)-2: Construction of a template plasmid using a flexible linker (FL: SNSSSVPGG (SEQ ID NO: 14)) The VPg segment from the LM14-2 plasmid was amplified by polymerase chain reaction (PCR) using a set of gene amplification primers: VPg_F (with an NdeI restriction enzyme site: ACGCCATATGGGCAAGAAAGGGAAGAACAAGTCC (SEQ ID NO: 18)) and VPg_R (with an EcoRI restriction enzyme site: GCTCGAATTCGACTCAAAGTTGAGTTTCTCATTGTAGTCAACAC (SEQ ID NO: 19)). The PCR product was then cloned into the plasmid pKN1-1 (a plasmid for expressing GFP-gp5f) (Patent Document 2) digested with NdeI and EcoRI.

[0128] As disclosed in Patent Document 2, the plasmid pKN1-1 was prepared by first amplifying the gene corresponding to residues 461 to 484 of the wac protein of T4 phage by PCR from the T4 phage genome and cloning it into pUC18 to obtain a gene encoding foldon. Subsequently, this plasmid was cleaved with restriction enzymes EcoRI and SalI and inserted into plasmid pET29b (Novagen) treated with EcoRI and XhoI to obtain plasmid pMTf1-3. Furthermore, the gene corresponding to residues 474 to 575 of gp5 of T4 phage was amplified by PCR from the T4 phage genome and cloned into pUC18 to obtain a gene encoding gp5. Subsequently, this plasmid was cleaved with restriction enzymes EcoRI and SalI and inserted into the aforementioned plasmid pMTf1-3 treated with EcoRI and XhoI to obtain plasmid pKA176. In addition, the GFP expression vector provided by Takahashi of Gunma University was cleaved with the restriction enzymes NdeI and EcoRI to obtain the gene encoding GFP, which was then inserted into the above-mentioned plasmid pKA176 that had been treated with the restriction enzymes NdeI and EcoRI.

[0129] The cloned gene fragment was introduced into competent cells of Escherichia coli BL21 (DE3) and confirmed by DNA sequencing, which confirmed the presence of the plasmid construct "PN-FL-VPg" of PN and VPg separated by a flexible linker (SNSSSVPGG: SEQ ID NO: 15).

[0130] (b)-3: Production of an aggregate using the sFL / sRL linker and confirmation of its contents The "short rigid linker" (sRL:PAPAP) of SEQ ID NO: 16 was used as the linker L. １ A set of gene amplification primers VPgPA-F (with XhoI restriction site: CCGGCTCCGGCCCCACTCGAGGGAAGCAATACAATATTTGTACG (SEQ ID NO: 20)) and VPgPA-R (CTCAAAGTTGAGTTTCTCATTGTAGTCAACAC (SEQ ID NO: 21)) and a "short flexible linker" (sFL:GGGGS) of SEQ ID NO: 15 were inserted into the linker L. １A set of gene amplification primers VPgGS-F (containing an XhoI restriction site: GGAGGCGGGGGTTCACTCGAGGGAAGCAATACAATATTTGTACG (SEQ ID NO: 22)) and VPgGS-R (same as the above VPgPA-R (SEQ ID NO: 21)) were used separately as gene amplification primers for inverted PCR using "PN-FL-VPg" as a template to generate the plasmid construct "PN-sRL-VPg" (L １ is a short rigid linker of SEQ ID NO: 16) and "PN-sFL-VPg" (L １ was constructed using a short flexible linker of SEQ ID NO: 15.

[0131] These two plasmids were then introduced into DH5α competent cells, and the resulting vectors were verified by DNA sequencing, followed by expression of PN-sRL-VPg and PN-sFL-VPg.

[0132] For the expressed "PN-sRL-VPg" and "PN-sFL-VPg," proteins were loaded onto a Ni affinity column and eluted with a higher imidazole gradient (20-500 mM imidazole) and 1 mM DTT. These fractions containing PN-VPg were combined, the volume reduced to 5 ml, and the buffer was changed overnight at 4°C using a dialysis membrane in a buffer containing 20 mM Tris-HCl, pH 8.0. The concentrate was filtered through a 0.2 μm filter and then loaded onto a HiTrap Q column and eluted with a step gradient of sodium chloride (0-1 M). PN-VPg (short linker)-containing fractions were identified by native PAGE and SDS-PAGE.

[0133] SDS-PAGE results showed a band with the calculated molecular weight of approximately 31 kDa, the same as that of the monomer of PN-VPg (sFL / sRL linker) (not shown). In addition, MALDI-TOF mass spectrometry results for PN-sRL-VPg showed signals for the monomer (Fig. 2(a): observed m / z: 31,955, calculated m / z: 31,514), trimer (Fig. 2(b): observed m / z: 95,257, calculated m / z: 94,542), and hexamer (trimer-dimer) (Fig. 2(c): observed m / z: 190,929, calculated m / z: 189,084), respectively. Furthermore, the MALDI-TOF mass spectrum of PN-sFL-VPg showed the PN-sFL-VPg monomer (Figure 3(a): observed m / z: 31266, calculated m / z: 31396), trimer (Figure 3(b): observed m / z: 93857, calculated m / z: 94188), and hexamer (trimer-dimer) (Figure 3(c): observed m / z: 187218, calculated m / z: 188376).

[0134] These results indicate that the molecular needles are essentially trimers and hexamers.

[0135] Example 1 Vaccine using a molecular needle carrying a test peptide presumed to be an RS virus MHC class II peptide In this example, a vaccine of the present invention was prepared using a molecular needle carrying, as an active ingredient, a peptide consisting of 68 amino acids in the RSV L protein as a test peptide, and its efficacy was investigated. As a result, it was revealed from its efficacy that this vaccine is vaccine 3 of the present invention, which acts not only on class II but also on class I.

[0136] (a) Production Example The RSV L protein is an RNA polymerase composed of 2166 amino acid residues (GenBank number: KM517573, SEQ ID NO: 23) and is a non-structural protein synthesized in infected cells (FIG. 4).

[0137]

[0138] The 68 amino acid residues (SEQ ID NO: 24) in the region from positions 451 to 518 of this L protein are predicted to be the epitope (i.e., MHC class II peptide) recognized by the MHC II receptor molecule of T cells, and also the region that binds to the P protein (phosphorylated protein: a nonstructural protein of RSV that controls phosphorylation).

[0139] The sequence of the 68 amino acid residues (SEQ ID NO: 24) is expressed in single-letter amino acid code as "KFYLLSSLSTLRGAFIYRIIKGFVNTYNRWPTLRNAIVLPLRWLNYYKLNTYPSLLEITENDLIILSG." A molecular needle carrying this as W shown in formula (1) was produced.

[0140] All reagents used in Example 1 were purchased from commercial suppliers and used without further purification. The gene fragment for the RSV L protein was obtained by amplifying a portion of the gene sequence encoding the L protein, excluding the terminal stop codon, from the genomic RNA of the RSV-Long strain, provided by Dr. Sawada of the Department of Viral Infection Control I, Institute of Life and Medical Sciences, Kitasato University. In addition, the gene fragment encoding the peptide in the region from positions 451 to 518 of the L protein was also obtained by amplifying the fragment.

[0141] A plasmid (pET29b(+) / F-PN) was prepared in which this 68 amino acid peptide was fused to PN at its C-terminus via GGGGS (SEQ ID NO: 15). This was then transformed into Escherichia coli (BL21 DE3) and expression was induced with IPTG.

[0142] In this example, the template plasmid construct "PN-FL-VPg" obtained in "(b)-3" above was used as a template, and the gene fragment encoding the MHC class I peptide-containing peptide was replaced with Vpg by the InFusion cloning method to construct the desired plasmid construct "pET29b(+) / Lpep-PN" (Figure 5).

[0143] Specifically, the peptide region from positions 451 to 518 of the L protein to which an ATG(M) had been added was amplified using InFusion RS-P sense 5'-GGAGATATACATATGaagttctatttattaagtag-3' (SEQ ID NO: 25) and InFusion RS-P antisense 5'-TGAACCCCCGCCTCCtcctgataaaataatcaaatc-3' (SEQ ID NO: 26), yielding an amplified product of the P region to which the underlined portion had been added. Separately, the entire vector portion excluding the VPg sequence was amplified by inverted PCR using the template plasmid construct "PN-FL-VPg" as a template and primers 5'-GGAGGCGGGGGTTCA-3' (SEQ ID NO: 27) and 5'-ATGTATATCTCCTTCTTAAAG-3' (SEQ ID NO: 28), to prepare a vector body. These two fragments were ligated by InFusion cloning to obtain the desired plasmid construct "pET29b(+) / Lpep-PN".

[0144] This plasmid was then introduced into DH5α competent cells, and the resulting vector was verified by DNA sequencing, followed by expression of Lpep-PN(+).

[0145] This expression was measured by culturing E. coli BL21 (DE3) carrying the plasmid "pET29b(+) / Lpep-PN" in LB medium containing 30 μg / ml of kanamycin at 37°C overnight. 600After the pH reached 0.8, 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and arabinose were added. 16-17 hours after the addition of IPTG and arabinose, the bacteria were harvested by centrifugation at 8,000 rpm for 5 minutes and incubated at 180 rpm at 20°C. The cell pellet was then suspended on ice in a buffer containing 100 mM Tris-HCl pH 8.0 and 5 mM imidazole with one tablet of cComplete, EDTA-free, and lysed by sonication. Cell debris was removed by centrifugation (17,500 rpm for 50 minutes). The supernatant was filtered through a 0.8 μm filter, applied to a Ni-affinity column, and eluted with the same buffer using a linear gradient of 5 mM to 250 mM imidazole at 4°C. Next, this Lpep-PN complex was dialyzed against 20 mM Tris / HCl pH 8.0, 0.2 M NaCl, and then further dialyzed against PBS and concentrated by ultrafiltration. During this process, Lpep-PN spontaneously formed into an complex containing trimers and / or hexamers. This complex was used in the immunoassay as the "Lpep-PN complex." Furthermore, as a control, a "peptide containing an MHC class I peptide consisting of 68 amino acid residues derived from the L protein" was prepared by gene amplification according to standard methods.

[0146] (b) Immunity Test (1) of Example 1 (Measurement of Antibody Titer) The above Lpep-PN aggregate group (PN(+)) and the above test peptide itself (hereinafter also referred to as LMHC peptide) consisting of 68 amino acid residues derived from the L protein (PN(-)) were intranasally inoculated into groups of three RS virus-susceptible cotton rats. All intranasal inoculations were performed by instilling and aspirating the vaccine solution into the nasal cavity. 300 μl (20 μg) of purified antigen per rat was aspirated into the nasal cavity under anesthesia. This intranasal inoculation was performed twice, one week apart after the initial inoculation. Blood samples were collected before the initial inoculation, one week after the initial inoculation (before the first booster inoculation), two weeks after the initial inoculation (before the second booster inoculation), and three weeks after the initial inoculation. The samples were reacted with RSV L protein, and serum IgG and IgA antibody titers against the RSV L protein were measured by ELISA.

[0147] The final booster vaccination was administered 7 weeks after the first vaccination.

[0148] For the ELISA, the antigen (RSV L protein) was diluted to 2 μg / mL with PBS(-) and added to a 96-well ELISA plate at 50 μL / well. The plate was incubated overnight at 4°C. The plate was washed three times with PBST (0.1% Tween 20 / PBS). 80 μL / well of PBSB (1% BSA / PBS) was added to each plate and incubated at room temperature for 2 hours for blocking. Test serum was then diluted with PBSB to prepare test samples (IgG detection: 5-fold serial dilutions from 10-31250-fold, IgA detection: 3-fold serial dilutions from 10-2430-fold). The PBSB in the plate was discarded, and 50 μL / well of each test sample was added to the plate. After incubation at room temperature for 2 hours, the plate was washed five times with PBST. 50 μL / well of HRP substrate solution was added to the plate, and the plate was incubated at room temperature in the dark until color development was confirmed. 25 μL / well of 2 M sulfuric acid was added to the plate to stop the reaction, and the absorbance at 490 nm was measured.

[0149] The results after 3 weeks (3W) are shown in Figure 6 (IgA) and Figure 7 (IgG). In both figures, the vertical axis represents absorbance and the horizontal axis represents serum dilution. These figures show the results for each individual cotton rat tested. These results show that direct immunization with the LMHC peptide did not increase the absorbance, which indicates antibody titers for both IgA and IgG, whereas immunization with the test peptide as an aggregate of molecular needles clearly increased these values, indicating significant induction of humoral immunity. Moreover, it is noteworthy that such a significant increase in antibody titer was achieved without the use of any adjuvant, demonstrating the feasibility of using this vaccine as an adjuvant-free vaccine.

[0150] Respiratory syncytial virus infection occurs when the virus is inhaled via droplet infection. Because the primary routes of infection are the oral cavity and nasal cavity, intranasal inoculation directly penetrates the cell membrane of the aggregates and introduces them into nasal mucosal cells, inducing humoral immunity within the nasal cavity and eliciting protective immunity against RS virus. Thus, it was demonstrated that intranasal inoculation of molecular needles carrying the test peptide as an immunogen induces local immunity. In particular, the induction of IgA against the L protein, a nonstructural protein of RS virus, suggests that IgA produced in the blood may bind to the L protein and inhibit its function when it passes through cells and is secreted into the mucosal layer. Furthermore, the region to which the induced antibody binds is also the region that binds to the P protein to exert L protein function. Therefore, inhibiting the formation of the replication complex between the L protein and the P protein may more efficiently inhibit viral replication and proliferation.

[0151] (c) Immunization Test (2) of Example 1 (Effect of Inhibiting Pulmonary Inflammation) In this test, a direct investigation was conducted on the effect of inhibiting pulmonary inflammation caused by RS virus sensitization in RS virus-susceptible cotton rats that had been immunized with molecular needles carrying peptides containing the test peptide.

[0152] Seven weeks after the initial inoculation and two weeks after the final booster inoculation in the above-mentioned immunization test (1) (i.e., nine weeks after the initial inoculation), the test cotton rats were infected with RS virus (Long strain). The infective dose of RS virus was 2 × 10 ５ The infection was carried out in the same manner as vaccination (intranasal inoculation) at PFU / mL. During this infection, a non-immunized, infected group of rats (3 rats) was added as a positive control.

[0153] Four days after the infection, lung tissues from the test rats were collected, and the amount of RS virus infection in the lungs was measured by removing the lungs, which are the target tissue for infection, suspending them in culture medium using a homogenizer to release the virus, and determining the amount of infectious virus contained in 50 mg of lung tissue using a plaque assay.

[0154] The results are shown in Figure 8. In Figure 8, the horizontal axis indicates "Not infected" for the negative control (n = 1), "Lpep-PN(+)" for the Lpep-PN aggregate group (n = 3), "Lpep-PN(-)" for the LMHC peptide group (n = 3), and "Not immunized" for the positive control (n = 3). The vertical axis indicates the RS virus titer (PFU / 50 mg of lung tissue) in the recovered lung tissue. As shown in Figure 8, rats administered with the Lpep-PN aggregate exhibited a significant suppression of RS virus infection by approximately 65% ​​compared to RS virus-infected rats (positive control), with a significant difference (P < 0.0001).

[0155] This remarkable result suggests that the test peptide supported on the molecular needle elicited not only the humoral immunity described above but also cellular immunity, resulting in extremely effective suppression of RS virus levels in the lungs. Therefore, the test peptide, which was initially assumed to contain an MHC class II recognition motif, likely also contains an MHC class I recognition motif. Such a strong suppression of virus levels cannot be explained solely by the humoral immune response mediated by MHC class II, and is thought to be due to the elimination of virus-infected cells by the induction of cellular immunity.

[0156] Example 2: Study on the novel coronavirus (1) Overview of the novel coronavirus The novel coronavirus (SARS-CoV-2) (hereinafter also referred to as the novel coronavirus) belongs to antigenic group 2 (betacoronavirus) of the genus Coronavirus, which includes the SARS coronavirus, the pathogen of SARS (severe acute respiratory syndrome), the human coronavirus OC43 strain that causes upper respiratory tract inflammation in humans, and the human coronavirus HKU1 that also causes lower respiratory tract inflammation, as well as those that infect mice, cattle, pigs, etc.

[0157] Similar to other coronaviruses, the novel coronavirus is spherical, measuring approximately 100 nm in diameter, with petal-like spikes that are thin at the base and swollen at the tip. The structural proteins of the novel coronavirus are the S (spike) protein, M (membrane) protein, and E (envelope) protein in the envelope. The S protein is a glycoprotein that forms a single petal-like spike as a trimer. It has the ability to adsorb to the host cell's viral receptor (angiotensin-converting enzyme II (ACE2)) and to fuse with the membrane via the action of serine protease (TMPRSS2). It also serves as a neutralizing epitope and a T cell epitope, serving as a target for the host immune response. The M protein and E protein are also glycoproteins, mostly located within the lipid bilayer, and play an important role in viral particle formation.

[0158] The N (nucleocapsid) protein is an RNA-binding phosphorylated protein that binds to viral genome RNA to form a nucleocapsid and is involved in RNA replication, transcription, and translation.

[0159] The novel coronavirus genome is also a positive-sense single-stranded RNA that functions as mRNA and is infectious. At least like the SARS coronavirus, the genome has a cap structure at the 5' end and a poly(A) at the 3' end, and the 5' end contains a leader sequence and untranslated region that regulate gene replication and transcription. Downstream of this are nonstructural protein genes that encode enzymes (replicases) essential for viral replication, such as RNA polymerase and protease, as well as structural genes that encode the S, E, M, and N proteins mentioned above.

[0160] Specifically, the S protein is composed of 1,273 amino acids and has an SS (signal sequence), NTD (N-terminal domain), RBD (receptor-binding domain), SD1 (subdomain 1), SD2 (subdomain 2), S1 / S2 (S1 / S2 protease cleavage site), S2' (S2' protease cleavage site), FP (fusion peptide), HR1 (heptad repeat 1), CH (central helix), CD (connector domain), HR2 (heptad repeat 2), TM (transmembrane domain), and CT (cytoplasmic tail). RBD is structured as a trimer consisting of two down protomers and one up protomer (Non-Patent Document 1, Non-Patent Document 2, etc.).

[0161] Nonstructural proteins are not proteins that form the novel coronavirus particle; rather, they are proteins involved in viral replication and proliferation. They are synthesized intracellularly through a series of viral protein expressions that occur when the virus attaches to and invades a cell and introduces the viral genome into the cell. Novel coronavirus protein synthesis is achieved by translation of only the ORF at the 5' end of each mRNA. Between the ORFs of the genome RNA (mRNA-1) and mRNA-2, there are two large ORFs, ORF1a and ORF1b. Protein 1a and protein 1b are translated from these ORFs, resulting in two proteins, 1a and 1a+1b. Protein 1a is cleaved into the nonstructural proteins NSP-1 through NSP-11 by its own proteolytic enzymes, NSP-3 (non-structural protein-3: papain-like protease) and NSP-5 (main protease). Furthermore, 1a+1b is cleaved during or after translation into nonstructural proteins nsp-1 to nsp-10, as well as nsp-12 to nsp-16. RNA-dependent RNA polymerase (nsp-12) and helicase (nsp-13) are produced as cleavage products from the 1b region. Many of these nonstructural proteins are essential for viral proliferation. "ORF3a," "ORF6," "ORF7a," and "ORF8," which are used as peptides derived from nonstructural proteins in Table 1 below, are all open reading frames located at the most downstream position after 25,000 bases in the genome.

[0162] (2) Test Peptides Used in Example 2 In this Example 2, a conjugated protein carrying a peptide consisting of nine amino acid residues shown in Table 1 below as the test peptide (W) was produced in the same manner as in Example 1, and a molecular needle (hexamer) was prepared by assembling these conjugated proteins. The "base positions" in the table are indicated with reference to [GenBank accession No. MN908947]. With one exception, RBD (receptor binding domain protein) was used as the test protein. All reagents used in this Example 2 were purchased from commercial suppliers and used without further purification.

[0163] The RBD constitutes part of the S protein, one of the structural proteins of the novel coronavirus (SARS-CoV-2). The RBD protein is encoded within the novel coronavirus genome as a template sequence for the messenger RNA of the S protein.

[0164]

[0165] In Example 2, the internal sequence of the sequence "" (SEQ ID NO: 31) from 22491 to 23182 in the spike gene (S-gene) of MN908947 from 21563 to 25384 bases was used.

[0166] The above-mentioned gene fragment within the RBD ((SEQ ID NO: 32), corresponding to the amino acid sequence: MFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATV (SEQ ID NO: 33)) was obtained by RT-PCR amplification of the gene sequence of the S protein from the virus strain (LC630936) D614G (name changed from "KUH003 strain") isolated and cultured from an infected patient in the Department of Viral Infection Control, Omura Satoshi Memorial Institute, Kitasato University.For infusion cloning, the fragment was used to prepare a molecular needle by adding the ggagatatacatATG sequence (SEQ ID NO: 34) to the 5' primer for RT-PCR and the ggaggcgggggttca sequence (SEQ ID NO: 35) (corresponding to the GGGGS linker) to the 3' primer.

[0167] In Table 1 below, "S" in the upper left corner indicates that the peptide is derived from a structural protein (as mentioned above, RBD is a protein), and "NS" in the lower left corner indicates that the peptide is derived from a non-structural protein.

[0168]

[0169] (3) Immunity Test 1 Test system 1 in Example 2 is a test system for measuring weight changes and lung virus load after infection with the new coronavirus after a specified immunization was administered to Syrian hamsters.

[0170] (a) The following five groups of Syrian hamsters were prepared, each group consisting of three animals.

[0171] S-PN(+): A test vaccine administration group (Group 1) constituted as a molecular needle administration group carrying structural protein peptides, prepared by mixing molecular needles (hexamers) carrying 13 types of structural protein peptides and RBD shown in Table 1 in equal amounts by mass, with the molecular needles being formulated to be 40 μg / mouse (in 30 ml of physiological saline) in one administration unit. NS-PN(+): A test vaccine administration group (Group 2) constituted as a molecular needle administration group carrying nonstructural protein peptides, prepared by mixing molecular needles (hexamers) carrying 9 types of nonstructural protein peptides shown in Table 1 in equal amounts by mass, with the molecular needles being formulated to be 40 μg / mouse (in 30 ml of physiological saline) in one administration unit. - S-PN(+) & NS-PN(+): A test vaccine administration group (Group 3) consisting of a 40 μg mixture of molecular needles carrying the 13 structural protein peptides and RBD of the above S-PN(+) in equal amounts by mass, and a 40 μg mixture of molecular needles carrying the 9 non-structural protein peptides of NS-PN(+) in equal amounts by mass, totaling 80 μg, contained in 30 ml of saline, which served as one administration unit. - SARS-CoV-2 inf: SARS-CoV-2 infected group (Group 4). - NC: Unimmunized (uninfected) group.

[0172] (b) The novel coronavirus used in the infection process was KUH003 (LC630936) D614G (a virus strain isolated and cultured from an infected patient in the Department of Viral Infection Control, Omura Satoshi Memorial Institute, Kitasato University).

[0173] (c) Test System 1 In Immunization Test 1, molecular needles carrying each test peptide were intranasally inoculated into the first, second, and third Syrian hamster groups at the respective dosage units. All intranasal inoculations were performed by dripping and aspirating the vaccine liquid into the nasal cavity, with 300 μl (40 μg) per animal being aspirated into the nasal cavity under anesthesia. This intranasal inoculation was performed twice, one week apart after the initial inoculation, and blood samples were taken before the initial inoculation, one week after the initial inoculation (before the first booster inoculation), two weeks after the initial inoculation (before the second booster inoculation), and three weeks after the initial inoculation. Blood samples were then taken seven weeks after the initial inoculation, and a final booster inoculation was administered. Two weeks after this final booster inoculation, the test hamsters were infected with the novel coronavirus. The infectious dose of the novel coronavirus was 2 × 10 ５ The infection was carried out in the same manner as vaccination (intranasal inoculation) at PFU / mL. During this infection, a group of unimmunized (uninfected) hamsters was added as a positive control.

[0174] The test hamsters were weighed daily for four days after the infection, and changes in weight due to COVID-19 infection were recorded. The weight changes are shown in Figure 9. Furthermore, on the final day, the lung tissue of the test rats was collected, and the amount of COVID-19 infection in the lungs was determined by removing the lungs, which are the target tissue for infection, suspending them in medium using a homogenizer to release the virus, and then determining the number of COVID-19 infections in 50 mg of lung tissue using a plaque assay. The amount of COVID-19 infection in the lung tissue is shown in Figure 10.

[0175] In Figure 9, the horizontal axis is the number of days elapsed, and the vertical axis is the relative change in weight (initial measurement taken as 100), showing the average value within each group. Group 1 (S-PN(+)) and Group 2 (NS-PN(+)) showed no weight loss. Group 3 (S-PN(+) & NS-PN(+)) showed a slight weight gain. Group 4 showed a significant weight loss. This indicates that, in the case of infection with the new coronavirus, Group 4, which did nothing, lost weight due to loss of appetite and physical exhaustion, whereas Groups 1 and 2, which received only S-PN(+) or NS-PN(+), maintained their current physical condition, and Group 3, which received both S-PN(+) and NS-PN(+), maintained a nearly normal state of health.

[0176] In Figure 10, the vertical axis represents the titer of the novel coronavirus in the collected lung tissue (PFU / 50 mg of lung tissue: x 1000). The results show that the weight change results are directly reflected in the viral load. Specifically, in Group 2 (NS-PN(+)), the viral load was reduced by approximately 60%. In Group 1 (S-PN(+)), the viral load was reduced by approximately 80%. Furthermore, in Group 3 (S-PN(+) & NS-PN(+)), almost no virus was detected. The reason for the greater viral load reduction in Group 1 compared to Group 2 is thought to be due to the stronger cellular immunity in Group 1. In other words, most of the test peptides in Group 1 functioned as MHC class I peptides, which strongly elicit cellular immunity, whereas those in Group 2 functioned as class II peptides, which preferentially elicit humoral immunity (based on the results of Immunity Test 2, described below). In Group 3, where Class I and Class II worked effectively, the proliferation of the new coronavirus was observed to be almost completely suppressed.

[0177] (4) Immunity Test 2 In immunity test 2, the molecular needles carrying the test peptides shown in Table 1 were individually examined for the secretion of various cytokines or chemokines associated with immunization.

[0178] A test vaccine administration system was constructed with molecular needles carrying each of the 13 structural protein peptides and RBD shown in Table 1, and each of the 9 nonstructural protein peptides, with each molecular needle being administered at a dose of 20 μg / mouse (in 30 ml of physiological saline) for each of three mice in each group.

[0179] The administration schedule for the test vaccines was as follows: each group of mice was intranasally inoculated with a molecular needle carrying the corresponding test peptide, using the above-mentioned dosage unit. Specifically, all intranasal inoculations were performed by instilling and aspirating the vaccine solution into the nasal cavity, with 300 μl (20 μg) per mouse being aspirated into the nasal cavity under anesthesia. This intranasal inoculation was performed twice, one week apart after the initial inoculation. Blood samples were collected before the initial inoculation, one week after the initial inoculation (before the first booster inoculation), two weeks after the initial inoculation (before the second booster inoculation), and three weeks after the initial inoculation. Blood samples were then collected seven weeks after the initial inoculation, and the final booster inoculation was administered.

[0180] Two weeks after the booster vaccination, mice were stimulated with a virus-infected cell suspension. Four days later, exsanguination was performed on the mice, and their spleens were removed. Immunocompetent cells were extracted from the spleens. These cells were stimulated with a cell suspension infected with KUH003 (LC630936) D614G (a virus strain isolated and cultured from an infected patient at the Department of Viral Infection Control, Omura Satoshi Memorial Institute, Kitasato University). Cytokines and chemokines in the immunocompetent cells were quantified using Bioplex before and after stimulation. Each quantification value was averaged within each group. The ratio of the post-stimulation quantification value to the pre-stimulation quantification value (post-stimulation / pre-stimulation) was calculated for each detection item (type of cytokine or chemokine) and test peptide. Test peptides were evaluated to identify those that exhibited enhanced cytokine or chemokine secretion characteristic of MHC class I or class II upon viral stimulation. The evaluation criteria were as follows: for MHC class I, peptides with a ratio of the above quantitative values ​​of five or more of seven cytokines or chemokines, namely, IFN-γ, IL-2, IL-10, IL-12(p40), IL-12(p70), MIP-1a, and TNF-α, that exceeded 2 were evaluated as MHC class I peptides; for MHC class II, peptides with a ratio of the above quantitative values ​​of seven or more of nine cytokines, namely, IFN-γ, IL-1b, IL-4, IL-5, IL-6, IL-10, IL-12(p40), IL-12(p70), and TNF-α, that exceeded 2 were evaluated as MHC class II peptides.

[0181] As a result of the above test, the peptides and RBDs of sample numbers #1, #2, #3, and #4 were found to have primarily MHC class II properties. Specifically, the four peptide samples were evaluated as MHC class II peptides because they showed increases in cytokine and chemokine expression upon stimulation via MHC class II. Furthermore, #7, #9, #11, #12, #15, #16, #17, #18, #19, and #20 were evaluated as MHC class I peptides because they showed increases in cytokine expression upon stimulation via MHC class I.

[0182] The results of Immunoassay 2 above are also an example of the information acquisition method of the present invention, which targets the novel coronavirus. That is, through the above steps, the ratio of the quantitative values ​​after novel coronavirus infection was calculated for each test peptide, and the results were used to evaluate the MHC type of each test peptide, enabling the above-mentioned MHC class I and class II discrimination evaluation for each sample.

Claims

1. [A] An amino acid sequence represented by the following formula (1-2): W ２ -L １ -X ｎ -Y (1-2) [In the formula, W ２ represents the amino acid sequence of a peptide or protein derived from a pathogenic microorganism that is the test immunogen, and L １ represents a first linker sequence having 0-100 amino acids, X represents the amino acid sequence of SEQ ID NO: 1, Y represents the amino acid sequence of the cell introduction region, and n, which is the number of repetitions of X, is an integer of 1-10. And, The amino acid sequence of the cell introduction region Y is represented by the following formula (2): Y １ -L ２ -Y ２ -Y ３ () [In the formula, Y １ represents any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 2-5; ２ represents any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 6-9, and L ２ represents a second linker sequence having 0-30 amino acids, and Y ３ represents the amino acid sequence for modification, and Y ２ Or Y ３ may not exist.] The conjugated protein is represented by the formula: ｎ , Y １ , or Y ２ A trimer or hexamer of a composite protein which is allowed to include a modified amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by the formula (I); A test animal is immunized with the trimer or hexamer represented by [A], immunocompetent cells of the immunized test animal are separated from the body of the test animal, one or more physiologically active substances in the separated immunocompetent cells are quantified, the separated immunocompetent cells are then infected with a target pathogenic microorganism, the physiologically active substances in the immunocompetent cells after the infection are quantified, and the amount of each physiologically active substance before and after the infection, which is obtained from the first two quantitative values, is used as an index to determine the amount of the test immunogen W. ２ A method for obtaining information, comprising obtaining information regarding secretion of a physiologically active substance after immunization in a mammal.

2. The method for acquiring information according to claim 1 , wherein the physiologically active substance includes a cytokine or a chemokine.

3. Information regarding the secretion of physiologically active substances is ２ 3. The method for acquiring information according to claim 1 or 2, wherein the information is information regarding whether a virus matches MHC class I, or whether a virus matches MHC class II, or whether a virus matches both of these.

4. The method for obtaining information according to any one of claims 1 to 3, wherein the pathogenic microorganism is a virus.

5. The amino acid sequence of the following formula (1-1): W-L １ -X ｎ -Y (1-1) [Wherein, W represents an amino acid sequence of a peptide containing one or more peptides derived from a target pathogenic microorganism that is evaluated as an immunogen against the target pathogenic microorganism that matches MHC class I by the method for obtaining information according to claim 1, and L １ represents a first linker sequence having 0-100 amino acids, X represents the amino acid sequence of SEQ ID NO: 1, Y represents the amino acid sequence of the cell introduction region, and n, which is the number of repetitions of X, is an integer of 1-10. And, The amino acid sequence of the cell introduction region Y is represented by the following formula (2): Y １ -L ２ -Y ２ -Y ３ () [In the formula, Y １ represents any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 2-5; ２ represents any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 6-9, and L ２ represents a second linker sequence having 0-30 amino acids, and Y ３ represents the amino acid sequence for modification, and Y ２ Or Y ３ may not exist.] The conjugated protein is represented by the formula: ｎ , Y １ , or Y ２ a component vaccine comprising as an active ingredient a trimeric protein and / or a hexameric protein of a conjugated protein which may contain a modified amino acid sequence in which one or more amino acids of the amino acid sequence represented by the formula (I) are deleted, substituted or added; A method for administering a component vaccine, comprising administering the component vaccine to activate immune function in a recipient against a target pathogenic microorganism by an MHC class I-matched peptide.

6. The amino acid sequence of the following formula (1-1): W-L １ -X ｎ -Y (1-1) [Wherein, W represents an amino acid sequence of a peptide containing one or more peptides derived from a target pathogenic microorganism that is evaluated as an immunogen against the target pathogenic microorganism that matches MHC class II by the method for obtaining information according to claim 1, and L １ represents a first linker sequence having 0-100 amino acids, X represents the amino acid sequence of SEQ ID NO: 1, Y represents the amino acid sequence of the cell introduction region, and n, which is the number of repetitions of X, is an integer of 1-10. And, The amino acid sequence of the cell introduction region Y is represented by the following formula (2): Y １ -L ２ -Y ２ -Y ３ () [In the formula, Y １ represents any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 2-5; ２ represents any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 6-9, and L ２ represents a second linker sequence having 0-30 amino acids, and Y ３ represents the amino acid sequence for modification, and Y ２ Or Y ３ may not exist.] The conjugated protein is represented by the formula: ｎ , Y １ , or Y ２ a component vaccine comprising as an active ingredient a trimeric protein and / or a hexameric protein of a conjugated protein which may contain a modified amino acid sequence in which one or more amino acids of the amino acid sequence represented by the formula (I) are deleted, substituted or added; A method for administering a component vaccine, comprising administering the component vaccine to activate immune function in a recipient against a target pathogenic microorganism by an MHC class II-compatible peptide.

7. The amino acid sequence of the following formula (1-1): W-L １ -X ｎ -Y (1-1) [Wherein, W represents an amino acid sequence of a peptide containing one or more peptides derived from a target pathogenic microorganism that is evaluated as an immunogen against the target pathogenic microorganism that matches both MHC class I and MHC class II by the method for obtaining information according to claim 1, and L １ represents a first linker sequence having 0-100 amino acids, X represents the amino acid sequence of SEQ ID NO: 1, Y represents the amino acid sequence of the cell introduction region, and n, which is the number of repetitions of X, is an integer of 1-10. And, The amino acid sequence of the cell introduction region Y is represented by the following formula (2): Y １ -L ２ -Y ２ -Y ３ () [In the formula, Y １ represents any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 2-5; ２ represents any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 6-9, and L ２ represents a second linker sequence having 0-30 amino acids, and Y ３ represents the amino acid sequence for modification, and Y ２ Or Y ３ may not exist.] The conjugated protein is represented by the formula: ｎ , Y １ , or Y ２ a component vaccine comprising as an active ingredient a trimeric protein and / or a hexameric protein of a conjugated protein which may contain a modified amino acid sequence in which one or more amino acids of the amino acid sequence represented by the formula (I) are deleted, substituted or added; A method for administering a component vaccine, comprising administering the component vaccine to activate immune function in a recipient against a target pathogenic microorganism by MHC class I / MHC class II compatible peptides.

8. In the above formula, X ｎ , Y １ , or Y ２ In the modified amino acid sequence in which one or more amino acids are deleted, substituted or added, the amino acid modification in each amino acid sequence is ｎ is 8n or less, Y １ Within 30, and Y ２ The method for administering a component vaccine according to any one of claims 5 to 7, wherein the number of the component vaccines is 15 or less.

9. A method for administering a component vaccine according to any one of claims 5 to 8, wherein the immunogen W is a peptide linked via a linker.

10. The method for administering a component vaccine according to any one of claims 5 to 9, wherein the pathogenic microorganism is a virus.

11. A method for administering a component vaccine according to any one of claims 5 to 10, which is subcutaneous, intradermal, transdermal, mucosal or intramuscular administration.

12. A method for administering a component vaccine according to any one of claims 5 to 10, which is administered to the nasal mucosa, pharyngeal mucosa, respiratory tract mucosa, sublingual mucosa or bronchial mucosa.

13. A method for administering a component vaccine as described in claim 12, wherein the dosage form of the component vaccine is a spray, an aerosol or a capsule.