Vaccine composition comprising BP26 nanostructure, antigen polypeptide, and assembly-type protein-binding module

Abstract

The present invention relates to a fusion protein comprising a BP26 nanostructure, an antigen polypeptide and an assembly-type protein-binding module, and a vaccine composition using same. The vaccine composition, of the present invention, comprising an assembly-type protein-binding module, in addition to a BP26 nanostructure and an antigen polypeptide, has a remarkable effect of effectively generating antibodies in vivo, and thus is expected to be actively used for various medical and health enhancement purposes.

Description

Vaccine composition comprising BP26 nanostructures, antigen polypeptides, and assembled protein binding modules

[0001] The present invention relates to a fusion protein comprising a BP26 nanostructure, an antigen polypeptide, and an assembled protein binding module, and a vaccine composition using the same.

[0002] Vaccines are antigens administered to the body to artificially generate immunity against infectious diseases, serving as a safe and effective method for combating and eradicating infectious diseases. Vaccine development has progressed from using attenuated or dead pathogens—aiming to generate the necessary protective immune response while avoiding inherent risks by utilizing attenuated strains—to using smaller antigenic components of these pathogens. Conventional vaccine technology focused on effectively expressing the immunogenicity of antigenic components, which were previously limited to short or small peptides and proteins, in order to overcome the problem that most antigens used in vaccine compositions were too large to be stably delivered into the body. However, this raised another problem: vaccines could fail to exert sufficient efficacy due to antigenic variable pathogen responses, which evade the immune response induced by vaccination through changes in specific parts of the antigen.

[0003] Meanwhile, various nanomaterials can be used as antigen delivery carriers to induce humoral or cellular immunity for the development of anticancer and antiviral vaccines. In particular, naturally occurring self-assembling protein nanostructures, such as ferritin and heat shock proteins, can hold or display antigens and can be used as antigen delivery carriers. While such nanostructures with antigen display, self-assembly, symmetrical structure, and nano-size can mimic virus-like architecture, they are not effective in inducing antigen-specific immune responses without the additional use of an adjuvant to activate immunity. Accordingly, the inventors of the present invention have recently developed a nanostructure effective in both adjuvant and carrier roles by identifying that BP26, an outer membrane protein of Brucella, forms nanostructures through self-assembly and that these nanostructures can effectively deliver antigens.

[0004] Accordingly, the inventors of the present invention have developed a vaccine composition by further developing the conventionally developed BP26 nanostructure and including an assembled protein binding module so that the nanostructure can more effectively deliver antigenic components into the body. Since the vaccine composition comprising the BP26 nanostructure and antigen polypeptide of the present invention, in addition to the assembled protein binding module, has a significant effect in effectively generating antibodies in vivo, it is expected to be actively utilized for various medical and health function enhancement purposes.

[0005] One object of the present invention is to provide a fusion protein comprising a BP26 nanostructure, an antigen polypeptide, and an assembled protein binding module.

[0006] Another objective of the present invention is to provide a nanostructure comprising the above-mentioned fusion protein.

[0007] Another objective of the present invention is to provide a vaccine composition comprising the above-mentioned fusion protein, nanostructure, or a combination thereof.

[0008] Another objective of the present invention is to provide a nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein described above, a recombinant vector comprising the nucleic acid molecule, or a transformant comprising the vector.

[0009] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0010] Various embodiments described herein are described with reference to the drawings. In the following description, for a complete understanding of the invention, various specific details, such as specific forms, compositions, and processes, are described. However, specific embodiments may be practiced without one or more of these specific details, or in combination with other known methods and forms. In other examples, known processes and manufacturing techniques are not described as specific details so as not to unnecessarily obscure the invention. Reference throughout this specification to "one embodiment" or "an embodiment" means that the particular features, forms, compositions, or characteristics described in association with the embodiment are included in one or more embodiments of the invention. Accordingly, the context of "in one embodiment" or "an embodiment" expressed at various places throughout this specification does not necessarily represent the same embodiment of the invention. Additionally, particular features, forms, compositions, or characteristics may be combined in any suitable way in one or more embodiments.

[0011] Unless otherwise specifically defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.

[0012] The inventors of the present invention have advanced the conventionally developed BP26 nanostructure and, after continuing strenuous efforts to develop a vaccine composition that includes an assembled protein binding module so that the nanostructure can more effectively deliver an antigenic component into the body, have succeeded in producing a vaccine composition comprising a BP26 nanostructure, an antigen polypeptide, and an assembled protein binding module that has a significant effect in effectively generating antibodies in vivo.

[0013] According to one aspect of the present invention, the present invention provides a fusion protein comprising a BP26 nanostructure, an antigen polypeptide, and an assembled protein binding module.

[0014] In this specification, the term "BP26" refers to the outer membrane protein of Brucella and is also referred to as BP26 / OMP28. BP26 has self-assembling properties and, as a major immunodominant antigen, exhibits antigen carrier and adjuvant effects.

[0015] In one embodiment of the present invention, the BP26 comprises the amino acid sequence of SEQ ID NO. 1, but is not limited thereto.

[0016] In this specification, the term "amino acid" refers to the most basic building block of a protein molecule. Looking at the structure of an amino acid, an amino group (-NH2) and a carboxyl group (-COOH) are attached to one carbon atom, and a hydrogen and an R group are connected thereto.

[0017] In one embodiment of the present invention, the BP26 comprises a variant of the amino acid sequence of SEQ ID NO. 1.

[0018] For example, changes to the amino acid sequence of the polypeptide may be made to improve the biological properties of the above BP26. Such changes include, for example, deletion, insertion, and / or substitution of amino acid sequence residues of the polypeptide.

[0019] The above variant has "substantial similarity" to the original polypeptide, meaning that when the two polypeptide sequences are optimally aligned, such as by a program GAP or BESTFIT using default gap weights, they share at least about 90% sequence identity, more preferably at least about 95%, 98%, or 99% sequence identity.

[0020] Preferably, the positions of non-identical amino acid residues are differentiated by conservative amino acid substitution. The "conservative amino acid substitution" is in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitution does not substantially alter the functionality of the protein.

[0021] These amino acid variations are based on the relative similarities of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0022] In introducing mutations, the hydropathic index of the amino acid may be considered. Each amino acid is assigned a hydropathic index based on hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0023] The hydrophobic amino acid index is very important in conferring interactive biological functions of proteins. It is a known fact that similar biological activity can be achieved by substituting with amino acids having similar hydrophobic indices. When introducing a variation based on the hydrophobic index, the substitution is preferably made between amino acids exhibiting a difference in hydrophobic index within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

[0024] Meanwhile, it is also well known that substitution between amino acids having similar hydrophilicity values ​​results in proteins having uniform biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); aspalate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); Phenylalanine (-2.5); tryptophan (-3.4).

[0025] When introducing a variation by referring to the hydrophilicity value, substitution is made between amino acids that exhibit a difference in hydrophilicity value within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

[0026] Amino acid exchanges in proteins that do not change the overall activity of the molecule are known in the art (H. Neurath, RLHill, The Proteins, Academic Press, New York, 1979). The most common exchanges are exchanges between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0027] In one embodiment of the present invention, the BP26 polypeptide of the present invention comprises a sequence having at least 90% sequence homology (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97, 98%, or 99%) with respect to the amino acid sequence of SEQ ID NO. 1. All integers from 90% to 100% and decimals existing between them are included within the scope of the present invention with respect to % homology.

[0028] In one embodiment of the present invention, the antigen polypeptide included in the fusion protein is a pathogen-derived antigen or a tumor-derived antigen.

[0029] In one embodiment of the present invention, the fusion protein comprises one or more of the pathogen-derived antigen polypeptides or tumor-derived antigen polypeptides. For example, in the present embodiment, the fusion protein of the present invention comprises the Receptor Binding Domain (RBD) of the wild-type SARS-CoV-2 (hCoV-19 / Korea / KCDC06 / 2020) virus as an antigen polypeptide. However, this is merely an example, and the fusion protein of the present invention may comprise 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 2, 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 4 to 20, 4 to 18, 4 to 16, 4 to 14, 4 to 12, 4 to 10, 4 to 8, or 4 to 6 pathogen-derived antigen polypeptides or tumor-derived antigen polypeptides, but is not limited thereto. No, it will be obvious to those skilled in the art that a sufficient number can be selected to trigger an immune response.

[0030] The term "pathogen" in this specification refers to a microorganism that parasitizes a host and causes disease, examples of which include viruses, rickettsia, bacteria, fungi, and protozoa.

[0031] The terms “polypeptide” or “protein” as used herein interchangeably refer to polymers of amino acid residues. Additionally, polypeptides and proteins apply to amino acid polymers in which one or more amino acid residues correspond to naturally occurring amino acids, as well as to analogs or mimics of naturally occurring amino acid polymers. Furthermore, polypeptides and proteins may comprise amino acid polymers that have been modified to form glycoproteins, for example, by the addition of carbohydrate residues.

[0032] The polypeptide or protein of the present invention may be prepared by synthetic methods known in the art, for example, by synthesizing a recombinant protein by transforming a host cell with an expression vector containing a nucleic acid molecule expressing a protein, or by solid-phase synthesis techniques (Merrifield, J. Amer. Chem. Soc. 85:2149-54(1963); Stewart, et al., Solid Phase Peptide Synthesis, 2nd. ed., Pierce Chem. Co.: Rockford, 111(1984)).

[0033] The pathogen-derived antigen polypeptide of the present invention can serve as an antigen for antibody production, and any antigen polypeptide derived from a pathogen can be selected regardless of its type.

[0034] In one embodiment of the present invention, the pathogen is selected from the group consisting of viruses, bacteria, rickettsia, fungi, and protozoa, but is not limited thereto.

[0035] In one embodiment of the present invention, the virus is SARS-CoV-2, MERS-CoV, HPV (Human papillomavirus), HBV (hepatitis B virus), HCV (hepatitis C virus), HIV (human immunodeficiency virus), Zika virus, HPV, norovirus, rotavirus, influenza virus, parvovirus, adenovirus, HSV (herpes simplex virus), porcine reproductive and respiratory syndrome virus (PRRSV), swine fever virus, porcine circovirus (PCV), porcine epidemic diarrhea virus (PEDV), foot-and-mouth disease virus, Newcastle virus, Burna virus, or viral hemorrhagic septicemia virus (VHSV), but is not limited thereto.

[0036] In one embodiment of the present invention, the bacteria are Escherichia coli, Clostridium perfringens, Campylobacter fetus jejuni, Mycobacterium paratuberculosis, Mycobacterium bovis, Clostridium tetani, Salmonella gallinarum, Staphylococcus aureus, Mycoplasma gallisepticum, Haemophilus parasuis, Lawsonia intracellularis, Bordetella bronchiseptica, and Pasteurella multocida Or Rhodococcus equi, but not limited thereto.

[0037] In one embodiment of the present invention, the rickettsia is Rickettsia rickettsia, Orientia tsutsugamushi, Coxiella burnetii, Rickettsia prowazekii, or Rickettsia typhi, but is not limited thereto.

[0038] In one embodiment of the present invention, the fungus is Candida spp., Mucor spp., Rhizopus spp., or Aspergillus spp., but is not limited thereto.

[0039] The above protozoa are Cryptosporidium, Eimeria, Trypanosoma, Plasmodium, or Toxoplasma, but are not limited thereto.

[0040] In one embodiment of the present invention, the antigen polypeptide derived from the pathogen for each pathogen comprises, in the case of birds, the VP2, VP3, and VP4 proteins of Infectious bursal disease virus (IBV), or a combination thereof; glycoprotein B, glycoprotein I, glycoprotein D, glycoprotein E, and glycoprotein C of Infectious laryngotracheitis virus (ILTV), or a combination thereof; fusion protein (NDV-F) of Newcastle disease virus; HA (hemagglutinin), NA (neuraminidase), and M2e proteins of avian influenza virus, or a combination thereof; S1 and S2 proteins of Infectious bronchitis virus (IBV), or a combination thereof; and OMP (outer membrane protein) of Salmonella gallinarum. Includes, but is not limited to, Net-B, PFO protein of Clostridium perfringens, or combinations thereof.

[0041] In one embodiment of the present invention, the antigen polypeptide derived from the pathogen according to the pathogen includes, in the case of pigs, proteins VP2, VP4, VP6 of rotavirus, or combinations thereof; proteins VP1, VP2, VP3, VP4 of P1 protein of foot and mouth disease virus (FMDV), or combinations thereof; protein VP2 of parvovirus; proteins E, ORF3, protein M of porcine reproductive and respiratory syndrome virus (PRRSV), or combinations thereof; E1, E2 of classical swine fever virus (CSFV), or combinations thereof; capsid protein of circovirus type 2 (PCV2); proteins k88, k99, 987p of Escherichia coli, or combinations thereof; OMP H (outer membrane protein H) of Pasteurella multocida, etc., but is not limited thereto.

[0042] In one embodiment of the present invention, in the case of a dog, the antigen polypeptide derived from the pathogen according to the pathogen includes, but is not limited to, glycoprotein G, nucleoprotein N, phosphoprotein P, matrix protein M, RNA polymerase L, or a combination thereof of rabies virus; HA protein, F protein, or a combination thereof of canine distemper virus.

[0043] In one embodiment of the present invention, the pathogen-derived antigen polypeptide is a coronavirus-derived polypeptide.

[0044] The term "Coronavirus" in this specification refers to four genera (Alpha, Beta, Gamma, and Delta) of the Coronavirinae subfamily of the Coronaviridae phylum. These are RNA viruses with a gene size of 27 to 32 kb that are known to cause respiratory and digestive system infections in humans and animals. Infection occurs easily, mainly through mucosal transmission and droplet spread. Humans generally experience mild respiratory infections, though fatal infections can occur rarely; cattle and pigs may develop diarrhea, while chickens may develop respiratory diseases. Among coronaviruses, those that use humans as hosts are classified according to the classification in Table 1 below (Korea Centers for Disease Control and Prevention, 2020). Of the four genera, Alpha and Beta infect both humans and animals, while Gamma and Delta are reported to infect only animals.

[0045] Genus Human-Coronavirus Non-human Infectious Coronavirus Alpha Coronavirus 229, NL63 Porcine Epidemic Diarrhea Virus (PEDV), Transmissible Gastroenteritis Virus (TGEV), Canine Coronavirus (CCoV), Feline Coronavirus (FCoV), Miniopterus Bat Coronavirus 1, Miniopterus Bat Coronavirus HKU8, Rhinolophus Bat Coronavirus HKU2, Scotophilus Bat Coronavirus 512 Beta Coronavirus OC43, HKU1, SARS-CoV, SARS-CoV 2, MERS-CoV Porcine Hemagglutinating Encephalomyelitis Virus (PHEV), Bovine Coronavirus (BCoV), Equine Coronavirus (EqCoV), Murine Coronavirus (MuCoV), Tylonycteris bat coronavirus HKU4, Pipistrellus bat coronavirus HKU5, Rousettus bat coronavirus HKU9, Gamma coronavirus-Avian coronavirus, Beluga whale-coronavirus SW1, Delta coronavirus-Bulbul-coronavirus HKU11, Thrush-coronavirus HKU12, Munia-coronavirus HKU13

[0046] In particular, there are currently 7 types of coronaviruses that are infectious to humans, as shown in Table 2 below, which are divided into types that cause the common cold (229E, OC43, NL63, HKU1) and types that cause severe pneumonia (SARS-CoV, SARS-CoV2, MERS-CoV).

[0047] Virus Name | Genus | Host | Symptoms HCoV-229 (EA) | α-Human | Mild respiratory symptoms HCoV-NL6 (3) | α-Human | Mild respiratory symptoms SARS-CoV (Beta) | Severe respiratory symptoms MERS-CoV (Beta) | Severe respiratory symptoms HCoV-OC4 (3) | β-Human | Mild respiratory symptoms HCoV-HKU1 (Beta) | Pneumonia symptoms SARS-CoV-2 (Beta) | Mild respiratory symptoms; causes shortness of breath in severe cases

[0048] In the present invention, the "Beta coronavirus" is one of the four genera of coronaviruses of the Coronaviruse family and corresponds to a zoonotic disease. Examples of beta coronaviruses are known to include the Severe Acute Respiratory Syndrome virus (SARS; SARS-CoV), Severe Acute Respiratory Syndrome virus-2 (SARS-CoV-2), Middle East Respiratory Syndrome virus (MERS; MERS-CoV), human coronavirus OC43 (HCoV-OC43), or human coronavirus HKU1 (HCoV-HKU1).

[0049] In the present invention, the "Severe Acute Respiratory Syndrome virus-2 (SARS-CoV-2)" corresponds to an enveloped, positive-polar single-stranded RNA beta coronavirus belonging to the coronavirus family. Historically, coronaviruses that infect humans are severe common cold viruses, including hCoV-OC43, HKU, and 229E5. Sequence analysis of SARS-CoV-2 isolates reveals that the 30-kb genome is known to code for 14 open-reading frames (ORFs), and 13 ORFs at the 3' end of the viral genome are expressed from 9 predicted subgenomic RNAs, which are expressed from 4 structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N), and 9 putative addition elements. Here, the nucleocapsid protein includes N1, N2, and N3 fragments.

[0050] In one embodiment of the present invention, the coronavirus includes a variant that has undergone natural or artificial mutation.

[0051] In one embodiment of the present invention, the pathogen-derived antigen polypeptide is the RBD (receptor binding domain) of the spike protein that appears in SARS-CoV 2.

[0052] In one embodiment of the present invention, the tumor-derived antigen comprises a tumor-associated antigen (TAA) and a tumor-specific antigen (TSA).

[0053] The above tumor-associated antigen (TAA) is an autoantigen that is abnormally overexpressed in tumors. T cells that recognize autoantigens can be eliminated through central tolerance and peripheral tolerance mechanisms.

[0054] The above tumor-specific antigens (TSAs) include neoantigens resulting from nonsynonymous mutations that alter the amino acids of proteins and genetic alterations, as well as oncoviral antigens of viruses. The above neoantigens further include shared neoantigens and private neoantigens that differ from individual to individual.

[0055] More specifically, the above tumor-derived antigen is selected from the group consisting of M30, MAGE-1, MAGE-2, MAGE-3, MAGE-12, BAGE, GAGE, NY-ESO-1, tyrosinase, TRP-1, TRP-2, gp100, MART-1, MCIR, Ig idotype, CDK4, Caspase-B, beta-catenin, CLA, BCR / ABL, mutated p21 / ras, mutated p53, proteinase 3, WT1, MUC-1, Her2 / neu, PAP, PSA, PSMA, G250, HPV E6 / E7, EBV LMP2a, CEA, alpha-Fetoprotein, 5T4, onco-trophoblast glycoprotein, etc., but is not limited thereto, and those skilled in the art will easily understand that various antigens applicable to cancer vaccines in this field may be applied.

[0056] In one embodiment of the present invention, the tumor (or cancer) includes, but is not limited to, breast cancer, head and neck cancer, bladder cancer, stomach cancer, colorectal cancer, pancreatic cancer, lung cancer, melanoma, prostate cancer, kidney cancer, liver cancer, biliary tract cancer, cervical cancer, thyroid cancer, etc.

[0057] In one embodiment of the present invention, the assembled protein binding module comprising the fusion protein is a set consisting of a first peptid moiety that binds to a BP26 nanostructure; and a second peptid moiety that binds to an antigen polypeptide. The first peptid moiety and the second peptid moiety are assembled by an isopeptide bond.

[0058] Recently, several genetically-encoded systems have been developed that enable spontaneous or auxiliary amide bond formation. For example, SnoopTag is a peptide engineered to form a spontaneous and irreversible isopeptide bond when mixed with its protein partner, SnoopCatcher. Within the protein chain, components of assembled protein binding modules, such as SnoopTag and SnoopCatcher, can be designed to be positioned at various locations and are reactive under a wide range of pH, buffer, and temperature conditions. Although various sets of assembled protein binding modules via isopeptide bonding, as well as their variants and derivatives, have been used in vaccine development, they have so far been limited to the presentation of simple antigens.

[0059] In one embodiment of the present invention, the first peptide moiety and the second peptide moiety set constituting the assembled protein binding module are any one selected from the group consisting of the SnoopTag and SnoopCatcher set, SnoopTagJr and SnoopCatcher set, SnoopTagJr and DogTag set, RrgATag and RrgACatcher set, RrgATag2 and RrgACatcher set, DogTag and RrgACatcher set, SpyTag and SpyCatcher set, IsopepTag and Pilin-C set, IsopepTag-N and Pilin-C set, IsopepTag and Pilin-N set, IsopepTag-N and Pilin-N set, and PsCsTag and PsCsCatcher set, but are not limited thereto, and those skilled in the art will know of peptides engineered to form spontaneous and irreversible assembly by isopeptide bonds in the field You will easily understand that sets can be applied.

[0060] In one embodiment of the present invention, the first peptid moiety constituting the assembled protein binding module comprises a sequence having at least 90% sequence homology (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97, 98%, or 99%) with respect to the amino acid sequence of SEQ ID NO. 4, and the second peptid moiety comprises a sequence having at least 90% sequence homology (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97, 98%, or 99%) with respect to the amino acid sequence of SEQ ID NO. 5. All integers from 90% to 100% and decimals existing between them are included within the scope of the present invention with respect to % homology.

[0061] In one embodiment of the present invention, a linker (e.g., an amino acid linker) is additionally connected between the antigen polypeptide and the second peptide moiety.

[0062] The term "linker" in this specification refers to a peptide inserted between proteins to increase the structural flexibility of the proteins or enhance the activity of each protein when two proteins are linked to form a fusion protein. The linker is not limited to any specific type as long as it does not inhibit the activity of each protein being fused and does not cause an unnecessary immune response, but specifically, it may be a peptide linker composed of 1 to 20 amino acids, more specifically 1 to 6 amino acids.

[0063] A person skilled in the art would be aware that the production of fusion proteins typically involves the use of linkers between the functional moieties to be fused, and that there are different types of linkers with different characteristics, such as flexible amino acid linkers, inflexible linkers, and cleavable amino acid linkers. Linkers have been used to increase the expression level of fusion proteins, enhance biological activity, enable targeting, modify pharmacokinetics, or increase the stability and improve folding of fusion proteins.

[0064] Accordingly, according to a specific embodiment of the present invention, the fusion protein may include at least one linker, such as a flexible amino acid linker, a non-flexible linker, and a cleavable amino acid linker.

[0065] According to a specific embodiment of the present invention, the linker comprises the amino acid sequence of SEQ ID NO. 6, but is not limited thereto.

[0066] In one embodiment of the present invention, the antigen polypeptide is connected continuously or discontinuously within the fusion protein.

[0067] The present invention can regulate an immune response by controlling the number of antigen polypeptides of a fusion protein, and in this case, various variant antigen polypeptides can be simultaneously transported within a single fusion protein by a tandem repeat structure.

[0068] The term "tandem repeat structure" in this specification refers to a configuration in which two or more antigen polypeptides included in the fusion protein of the present invention are connected in series, either continuously or discontinuously by a linker.

[0069] According to an embodiment of the present invention, BP26 of the present invention has the characteristic of being capable of self-assembly as an octamer, and since one BP26-antigen polypeptide conjugate can be assembled into a hexadecameric hollow barrel structure, the fusion protein can self-assemble to form a nanostructure.

[0070] According to another aspect of the present invention, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein.

[0071] In this specification, the term "nucleic acids" has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acid molecules, include not only natural nucleotides but also analogues in which sugar or base sites are modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)). In one embodiment of the present invention, it is obvious to those skilled in the art that the nucleotide sequence encoding the fusion protein of the present invention is sufficient to be a nucleotide sequence encoding the amino acid sequence constituting the fusion protein, and is not limited to any specific nucleotide sequence. This is because even if a mutation in the nucleotide sequence occurs, there are cases where expressing the mutated nucleotide sequence as a protein does not bring about a change in the protein sequence. This is called codon degeneracy. Accordingly, the above nucleotide sequence includes a nucleotide sequence containing functionally equivalent codons or codons encoding the same amino acid (for example, due to the degeneracy of codons, there are six codons for arginine or serine), or codons encoding biologically equivalent amino acids.

[0072] Considering the variant having the aforementioned biological equivalence activity, the nucleic acid molecule of the present invention encoding the amino acid sequence constituting the fusion protein is interpreted to also include a sequence exhibiting substantial identity therewith. The above substantial identity is obtained by aligning the sequence of the present invention described above with any other sequence to correspond as much as possible, and analyzing the aligned sequence using an algorithm commonly used in the art.

[0073] At least 60% homology (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69%), more preferably at least 70% homology (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%), more preferably at least 80% homology (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%), most preferably 90% or more (e.g., 90%, 91%, 92%, 93%, 94%, 95%, It refers to a sequence exhibiting homology of 96%, 97%, 98%, 99%, or 100%. All integers between 70% and 100% and decimals in between are included within the scope of the present invention with respect to % homology.

[0074] Alignment methods for sequence comparison are known in the art. Various alignment methods and algorithms are cited in Smith and Waterman, Adv. Appl. Math. 2:482 (1981); Needleman and Wunsch, J. Mol. Bio.48:443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc. Acids Res. 16:10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8:155-65 (1992) and Pearson et al., Meth. Mol. It is disclosed in Biol.24:307-31(1994), but is not limited thereto.

[0075] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10(1990)) is accessible from sources such as the National Center for Biological Information (NBCI) and can be used online in conjunction with sequencing analysis programs such as blastp, blastn, blastx, tblastn, and tblastx. BLAST can be accessed through the BLAST page on the NCBI website. Methods for comparing sequence homology using this program can be found on the BLAST help page of the NCBI website.

[0076] According to another aspect of the present invention, the present invention provides a recombinant vector comprising a nucleic acid molecule encoding the fusion protein.

[0077] The term "vector" as used herein includes plasmid vectors; cosmid vectors; and viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors, as means for expressing a target gene in a host cell.

[0078] In one embodiment of the present invention, the nucleic acid molecule encoding the fusion protein in the vector of the present invention is operatively linked to the promoter of the vector.

[0079] In this specification, the term "operationally coupled" means a functional coupling between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, thereby allowing the regulatory sequence to regulate the transcription and / or translation of the other nucleic acid sequence.

[0080] The recombinant vector system of the present invention can be constructed through various methods known in the art, specific methods for which are disclosed in Sambrook et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

[0081] The vector of the present invention can typically be constructed as a vector for gene cloning or as a vector for protein expression. Additionally, the vector of the present invention can be constructed using prokaryotic or eukaryotic cells as a host.

[0082] For example, when the vector of the present invention is an expression vector and the host is a eukaryotic cell, a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter and human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter and Rhoese's sarcoma virus (RSV) promoter) may be used, and these generally have a polyadenylation sequence as a transcription termination sequence.

[0083] The vector of the present invention may be fused with other sequences to facilitate the purification of polypeptides or proteins expressed therefrom. Examples of sequences to be fused include glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6x His(hexahistidine; Quiagen, USA).

[0084] Meanwhile, the expression vector of the present invention includes antibiotic resistance genes commonly used in the art as selection markers, such as resistance genes for ampicillin, gentamicin, cabbageillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0085] According to another aspect of the present invention, the present invention provides a transformant comprising the vector.

[0086] The above-mentioned transgenic bodies include animals other than humans, animal cells, plant cells, microorganisms (strains), and host cells such as yeast.

[0087] Host cells capable of stably and continuously cloning and expressing the vector of the present invention are known in the art, and any host cell may be used.

[0088] For example, suitable eukaryotic host cells for the above vector include, but are not limited to, monkey kidney cells 7 (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell line, HuT 78 cells, and HEK-293 cells.

[0089] In this specification, the terms "transformed," "transduced," or "transfected" refer to the process in which exogenous nucleic acids are delivered or introduced into a host cell. "Transformed," "transduced," or "transfected" cells are cells that have been transformed, introduced, or transfected with exogenous nucleic acids, and said cells include said cells and progeny cells resulting from their passage culture.

[0090] The recombinant vector of the present invention can be introduced into a host cell or strain through transformation (or transfection). Any transformation method used in the present invention may be used and can be easily performed according to conventional methods in the art. Specifically, the recombinant vector can be introduced into a host cell using, but is not limited to, conventional bacterial transformation methods, CaCl2 precipitation methods, the Hanahan method which increases efficiency by using DMSO (Dimethyl sulfoxide) as a reducing agent in the CaCl2 method, electroporation, calcium phosphate precipitation methods, protoplasmic fusion methods, stirring methods using silicon carbide fibers, Agrobacterium-mediated transformation methods, PEG-mediated transformation methods, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation methods.

[0091] According to another aspect of the present invention, the present invention provides a nanostructure comprising the fusion protein.

[0092] In one embodiment of the present invention, the nanostructure comprises two or more of the fusion proteins.

[0093] In one embodiment of the present invention, the nanostructure comprises 2 to 20 of the fusion proteins. More specifically, the nanostructure comprises 8 or 16 of the fusion proteins.

[0094] In one embodiment of the present invention, the nanostructure comprises a sixteenth mer formed by combining two octamers formed by eight fusion proteins.

[0095] In one embodiment of the present invention, the nanostructure comprises a hollow structure. A hollow structure refers to a structure that is hollow inside, and according to an embodiment of the present invention, the nanostructure of the present invention exhibits a hollow barrel-shaped structure.

[0096] According to an embodiment of the present invention, the hollow structure of the nanostructure exhibits a smaller pore size as the fusion protein included in the nanostructure contains more antigen polypeptides.

[0097] In one embodiment of the present invention, the nanostructure displays an antigen polypeptide on its surface. According to an embodiment of the present invention, the nanostructure displays a receptor binding domain (RBD) of the SARS-CoV-2 (hCoV-19 / Korea / KCDC06 / 2020) virus on its surface.

[0098] Since the fusion protein forming the nanostructure is identical to the fusion protein containing BP26 and the antigen polypeptide, the description of the overlapping parts is omitted to avoid excessive complexity in the specification.

[0099] According to another aspect of the present invention, the present invention provides a vaccine composition comprising the fusion protein, the nanostructure, or a combination thereof.

[0100] In one embodiment of the present invention, the vaccine composition is a vaccine composition for preventing viruses selected from the group consisting of SARS-CoV-2, MERS-CoV, HPV (Human papillomavirus), HBV (hepatitis B virus), HCV (hepatitis C virus), HIV (human immunodeficiency virus), Zika virus, HPV, norovirus, rotavirus, influenza virus, HSV (herpes simplex virus), porcine genital and respiratory syndrome virus (PRRSV), swine cholera virus, porcine circovirus (PCV), porcine diarrhea virus (PEDV), foot-and-mouth disease virus, Newcastle virus, and viral hemorrhagic sepsis virus (VHSV), but is not limited thereto.

[0101] In one embodiment of the present invention, the vaccine composition is a vaccine composition for preventing infectious pathogen infections.

[0102] In a specific embodiment of the present invention, the vaccine composition comprises Escherichia coli, Clostridium perfringens, Campylobacter fetus jejuni, Mycobacterium paratuberculosis, Mycobacterium bovis, Clostridium tetani, Salmonella gallinarum, Staphylococcus aureus, Mycoplasma gallisepticum, Haemophilus parasuis, Lawsonia intracellularis, Bordetella bronchiseptica, and Pasteurella multocida The vaccine composition for preventing bacteria is selected from the group consisting of and Rhodococcus equi, but is not limited thereto.

[0103] In one embodiment of the present invention, the vaccine composition is a rickettsia preventive vaccine composition selected from the group consisting of Rickettsia rickettsia, Orientia tsutsugamushi, Coxiella burnetii, Rickettsia prowazekii, or Rickettsia typhi, but is not limited thereto.

[0104] In one embodiment of the present invention, the vaccine composition is a fungal preventive vaccine composition selected from the group consisting of Candida spp., Mucor spp., Rhizopus spp., and Aspergillus spp., but is not limited thereto.

[0105] In one embodiment of the present invention, the vaccine composition is a vaccine composition for preventing protozoa selected from the group consisting of Cryptosporidium, Eimeria, Trypanosoma, Plasmodium, and Toxoplasma, but is not limited thereto.

[0106] According to an embodiment of the present invention, the vaccine composition is a vaccine composition for preventing coronavirus.

[0107] In one embodiment of the present invention, the vaccine composition is a vaccine composition for cancer vaccines.

[0108] The cancer vaccine of the present invention may be used to induce an immune response in mammals against cancer or tumors in mammals or subjects requiring it. The induced immune response may prevent or treat cancer or tumor growth.

[0109] The induced immune response can prevent and / or reduce the metastasis of cancer or tumor cells.

[0110] Depending on the antigen used in the vaccine, the above cancers include, but are not limited to, breast cancer, head and neck cancer, bladder cancer, stomach cancer, colorectal cancer, pancreatic cancer, lung cancer, melanoma, prostate cancer, kidney cancer, skin cancer, liver cancer, biliary tract cancer, cervical cancer, thyroid cancer, blood cancer (e.g., leukemia, lymphoma, multiple myeloma), etc.

[0111] In one embodiment of the present invention, the composition additionally comprises an adjuvant.

[0112] The term "adjuvant" in this specification refers to an additive substance intended to enhance an immune response, and is generally used together with an antigen for antibody production and immune enhancement. Specifically, the adjuvant is selected from the group consisting of alum, Freund complete adjuvant, Freund incomplete adjuvant, LPS, poly IC, poly AU, lysolecithin, fluron polyol, polyanionic, oil or hydrocarbon emulsion, keyhollympet hemocyanin, and dinitrophenol, but is not limited thereto.

[0113] In one embodiment of the present invention, the adjuvant is an immunostimulating single-chain or double-chain oligonucleotide, an immunostimulating small molecule compound, or a combination thereof.

[0114] In one embodiment of the present invention, the immunostimulating single-stranded or double-stranded oligonucleotides are known as useful adjuvants. They often contain a CpG motif (a dinucleotide sequence containing unmethylated cytosine linked to guanosine). Oligonucleotides containing a TpG motif, a palindrome sequence, a plurality of consecutive thymidine nucleotides (e.g., TTTT), a plurality of consecutive cytosine nucleotides (e.g., CCCC), or a poly(dG) sequence are also known adjuvants, such as double-stranded RNA. Any one of the various immunostimulating oligonucleotides may be used with the present invention without limitation.

[0115] The above oligonucleotide typically has 10 to 100 nucleotides, for example, 15 to 50 nucleotides, 20 to 30 nucleotides, or 25 to 28 nucleotides. It is typically a single strand.

[0116] The above oligonucleotides may comprise only natural nucleotides, only non-natural nucleotides, or a mixture of both. For example, the above oligonucleotides may comprise one or more phosphorothioate bonds and / or one or more 2'-O-methyl modifications.

[0117] In a specific embodiment of the present invention, the single-strand or double-strand oligonucleotide is a CpG oligonucleotide, a STING-active oligonucleotide, or a combination thereof.

[0118] In this specification, the term “Stimulator of Interferon Genes (STING)" refers to a stimulator of interferon genes (STING), a molecule that plays a major role in the innate immune response. STING comprises five putative transmembrane (TM) regions located primarily within the endoplasmic reticulum (ER) and can induce type I IFG and activate the NF-kB and IRF3 transcriptional pathways, which exert a potent antiviral state after expression (see U.S. Patent Application Serial No. 13 / 057,662 and PCT / US2009 / 052767). Loss of STING reduces the ability of polyIC to activate type I IFN and provides mouse embryonic fibroblasts (- / - MEF) susceptible to vesicular stomatitis virus (VSV) infection, lacking STING generated by targeted homologous recombination. In the absence of STING, DNA-mediated type I IFN responses are inhibited, indicating that STING may play a crucial role in recognizing DNA from viruses, bacteria, and other pathogens capable of infecting cells. Yeast double hybridization and co-immunoprecipitation studies revealed that STING interacts with RIG-I and Ssr2 / TRAPβ (members of the translocon-associated protein (TRAP) complex required for protein transport across the post-translational ER membrane). RNAi removal of TRAPβ inhibited STING function and disrupted the production of type I IFN in response to polyIC. Further experiments demonstrated that STING itself binds to nucleic acids, including single and double-stranded DNA from pathogens or apoptotic DNA, and plays a central role in regulating pro-inflammatory gene expression in inflammatory conditions such as DNA-mediated arthritis and cancer.Various novel methods for upregulating STING expression or function and various novel compositions for upregulating STING expression or function are described herein, along with further characterization of other cellular molecules interacting with STING. These findings enable the design of novel adjuvants, vaccines, and therapeutic regimens for regulating the immune system and other systems.

[0119] The STING-activating oligonucleotide may be a nucleic acid molecule that binds STING function to STING. The STING-binding nucleic acid molecule may be single-stranded DNA with a length of 40 to 150 base pairs or double-stranded DNA with a length of 40 to 150, 60 to 120, 80 to 100, or 85 to 95 base pairs or more. The STING-binding nucleic acid molecule may be nuclease-resistant, for example, prepared from nuclease-resistant nucleotides. The STING-binding nucleic acid molecule may also bind to a molecule that promotes transmembrane transport. In this method, the disease or disorder may be a DNA-dependent inflammatory disease. Additionally, a method for treating cancer in a subject having a cancerous tumor infiltrated by inflammatory immune cells is described herein. This method may include the step of administering any amount of a pharmaceutical composition comprising an agent that downregulates the function or expression of STING and a pharmaceutically acceptable carrier to the subject.

[0120] More specifically, the above oligonucleotide is an antisense oligonucleotide, a CpG oligonucleotide, or a combination thereof.

[0121] In this specification, the term CpG oligonucleotide (CpG oligodeoxynucleotide, or CpG oligodeoxynucleotide, CpG ODN) refers to a short single-stranded synthetic DNA molecule comprising unmethylated cytosine triphosphate deoxynucleotide ("C") and guanine triphosphate deoxynucleotide ("G"), and is known as an immunostimulant. When the CpG is included as a component of the nano vaccine of the present invention, it acts as an adjuvant that enhances the immune response of dendritic cells.

[0122] The above-mentioned immunostimulating small molecule compounds are also called small molecule adjuvants, and include synthetic small molecule adjuvants and natural small molecule adjuvants. Examples of the above-mentioned immunostimulating small molecule compounds or small molecule adjuvants include, but are not limited to, monophosphoryl lipid A, Muramyl dipeptide, Bryostatin-1, Mannide monooleate (Montanide ISA 720), Squalene, QS21, Bis-(3′,5′)-cyclic dimeric guanosine monophosphate, PAM2CSK4, PAM3CSK4, Imiquimod, Resiquimod, Gardiquimod, Cl075, Cl097, Levamisole, 48 / 80, Bupivacaine, Isatoribine, Bestatin, Sm360320, and Loxoribine. Regarding small molecule adjuvants, Flower DR et al. It is described in (Expert Opin Drug Discov. 2012 Sep;7(9):807-17.).

[0123] The vaccine composition of the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent.

[0124] In this specification, the term “pharmaceuticalally acceptable” refers to characteristics and / or substances that are acceptable to patients from a pharmacological / toxicological perspective and acceptable to dispensing pharmacists from a physical / chemical perspective regarding composition, formulation, safety, patient tolerance, and bioavailability. “Pharmaceutically acceptable carrier” refers to a medium that does not interfere with the effect of the biological activity of the active ingredient(s) and is non-toxic to the host upon administration. Carriers suitable for the vaccine are known to those skilled in the art and include, but are not limited to, proteins, sugars, etc. The carriers may be aqueous solutions or non-aqueous solutions, suspensions, and emulsions. Non-aqueous carriers include propylene glycol, polyethylene glycol, edible oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions, including saline solution and buffer medium.

[0125] Suitable dosages of the vaccine composition of the present invention can be prescribed in various ways depending on factors such as the species of the individual, breed of the individual, method of formulation, method of administration, age, body weight, sex, vaccination history, food, time of administration, route of administration, excretion rate, and response responsiveness.

[0126] For example, the dosage of the vaccine composition of the present invention may be 0.1 ml to 10 ml. The dosage of the vaccine composition of the present invention may be 0.1 ml to 0.5 ml for avian species, more specifically 0.3 ml to 0.5 ml. The dosage of the vaccine composition of the present invention may be 0.2 ml to 3.0 ml for cat, dog, and horse species, more specifically 0.3 ml to 2.0 ml, most specifically 0.5 ml to 1.0 ml. For cattle and pig species, the dosage of the vaccine composition of the present invention may be 0.2 ml to 5.0 ml, more specifically 0.3 ml to 3.0 ml, and more specifically 0.5 ml to 2.0 ml.

[0127] In addition, the amount of antigen in the administered dose of the vaccine composition may be 1 to 100 μg, 5 to 50 μg, 5 to 30 μg, 5 to 25 μg, 10 to 50 μg, 10 to 40 μg, 10 to 30 μg, or 10 to 25 μg. According to an embodiment of the present invention, the amount of antigen in the administered dose of the vaccine composition is 10 to 30 μg.

[0128] The subjects of application of the present invention may be any animal, specifically mammals, e.g., humans, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, monkeys, cattle, horses, pigs; and livestock, including birds, e.g., chickens, ducks, turkeys. The most preferred subjects are livestock or humans.

[0129] The vaccine composition may be freeze-dried or formulated as a liquid preparation by any means suitable in the art. Non-limiting examples of preparations in liquid form include solutions, suspensions, syrups, slurries, and emulsions. Suitable liquid carriers include any suitable organic or inorganic solvent, e.g., water, alcohol, saline solution, buffered saline solution, physiological saline solution, dextrose solution, water-propylene glycol solution, etc., and are preferably present in a sterile form.

[0130] The vaccine composition may be formulated in a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino group of the active polypeptide), which are formed with inorganic acids, e.g., hydrochloric acid or phosphoric acid, or organic acids, e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Additionally, salts formed from the free carboxyl group may be derived from inorganic bases, e.g., sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases, e.g., isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0131] The vaccine composition is preferably formulated for inoculation or injection into a subject. For injection, the vaccine composition of the present invention may be formulated in an aqueous solution, e.g., water or alcohol, or in a physiologically suitable buffer, e.g., Hanks' solution, Ringer's solution, or physiological saline buffer. The solution may include a formulation agent, e.g., a suspending agent, a preservative, a stabilizer, and / or a dispersant. Additionally, the injection formulation may be prepared as a solid form formulation that is converted into a liquid form formulation suitable for injection immediately before use, e.g., by reconstituting with a suitable vehicle, e.g., sterile water, saline solution, or alcohol, before use.

[0132] Additionally, the vaccine composition may be formulated as a delayed-release vehicle or a depot formulation. These long-acting formulations may be administered by inoculation or implantation (e.g., subcutaneous or intramuscular) or by injection. Thus, for example, the vaccine composition may be formulated with a suitable polymeric or hydrophobic material (e.g., as an emulsion in an acceptable oil) or an ion exchange resin, or with a sparingly soluble derivative, e.g., a sparingly soluble salt. Liposomes and emulsions are widely known examples of delivery vehicles suitable for use as carriers.

[0133] The vaccine composition may be administered by infusion or injection (e.g., intravenously, intramuscularly, intradermally, subcutaneously, intrathecally, intraduodenly, intraperitoneally, etc.). Additionally, the vaccine composition may be administered nasally, vaginally, rectalally, orally, or transdermally. Furthermore, the vaccine composition may be administered by a "needle-free" delivery system. Preferably, the composition is administered by intradermal injection. Administration may be performed under the direction of a physician or medical assistant.

[0134] The injection may be divided into several injections, and such split vaccinations are preferably administered substantially simultaneously. When administered as split vaccinations, the dose of the immunogen is preferably, but not necessarily, allocated equally to each separate injection. If an adjuvant is present in the vaccine composition, the dose of the adjuvant is preferably, but not necessarily, allocated equally to each separate injection. The separate injections for split vaccination are administered in some forms substantially adjacent to each other on the patient's body. In some forms, the injections are administered at least about 1 cm apart from each other on the body. In some forms, the injections are administered at least about 2.5 cm apart from each other on the body. In some forms, the injections are administered at least about 5 cm apart from each other on the body. In some forms, the injections are administered at least about 10 cm apart from each other on the body. In some forms, the injections are administered more than 10 cm apart from each other on the body, for example, at least about 12.5, 15, 17.5, 20 cm or more apart. The primary vaccination injection and the booster injection may be administered as split doses, as described and exemplified herein.

[0135] Various alternative pharmaceutical delivery systems may be used. Non-limiting examples of such systems include liposomes and emulsions. Certain organic solvents, for example, dimethyl sulfoxide, may also be used. Additionally, the vaccine composition may be delivered using a delayed-release system, for example, a semipermeable matrix of a solid polymer containing the therapeutic agent. Various available delayed-release materials are widely known to those skilled in the art. Delayed-release capsules, depending on their chemical properties, may release the vaccine composition over a range of days, weeks, or months.

[0136] To prevent the recurrence of cancer in a patient in a state of cancer remission, a therapeutically effective amount of a vaccine composition is administered to the subject. The therapeutically effective amount is, when measured by any means suitable in the art, the tumor-derived antigen-specific cytotoxic T-lymphocytes (CD8) in the patient + It will provide a clinically significant increase in the number of ) and a clinically significant increase in the cytotoxic T-lymphocyte response to the antigen. Generally, in patients, a therapeutically effective amount of the vaccine composition will destroy remaining microscopic disease, thereby significantly reducing or eliminating the risk of cancer recurrence in patients.

[0137] The effective dose of the vaccine composition may depend on many variables, including but not limited to race, breed, dimensions, height, weight, age, the patient's overall health status, the type of formulation, the mode or method of administration, or the presence or absence of risk factors that significantly increase the likelihood of cancer recurrence in the patient. These risk factors include, but not limited to, the type of surgery, the status and number of lymph nodes, the size of the tumor, the histological grade of the tumor, the presence or absence of hormone receptors (estrogen and progesterone receptors), HER2 / neu expression, lymphovascular invasion, and genetic predisposition (e.g., BRCA 1 and 2). In some preferred embodiments, the effective dose depends on whether the patient is lymph node-positive or lymph node-negative, and if the patient is lymph node-positive, it depends on the number and extent of positive lymph nodes. In all cases, the appropriate effective dose may be determined by a person skilled in the art using generally conventional optimization techniques, the proficient and knowledgeable judgment of an expert, and other factors obvious to a person skilled in the art. Preferably, the therapeutically effective amount of the vaccine composition described herein will provide therapeutic benefits without causing substantial toxicity to the subject.

[0138] The toxicity and therapeutic efficiency of a vaccine composition, for example, LD 50 (lethal dose for 50% of the population) and ED50 It can be measured by standard pharmaceutical procedures in cell cultures or experimental animals to determine (the therapeutically effective dose for 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, which is the LD50. 50 / ED 50 It can be expressed as a ratio. A vaccine composition exhibiting a large therapeutic index is desirable. Data obtained from cell culture assays and animal studies can be used to formulate a range of doses for use in patients. The dose of such a vaccine composition is preferably an ED with very low or no toxicity. 50 It falls within a certain range of circulating concentrations including. The dosage may vary within this range depending on the dosage form used and the route of administration utilized.

[0139] Toxicity information can be used to more accurately determine the useful dose for specific subjects, e.g., humans. Treatment may be terminated, discontinued, or adjusted due to toxicity or organ dysfunction, and treatment may be adjusted as necessary if the clinical response is not suitable for improving the response. In the prevention of recurrent cancer, the dosage amount will vary depending on the severity of the patient's condition, the relative risk of recurrence, or the route of administration, among other factors. The severity of the patient's condition may be assessed, for example, in part by standard prognostic assessment methods.

[0140] The vaccine composition may be administered to a patient at any suitable schedule to induce and / or support a cytotoxic T lymphocyte response, in order to induce and / or support protective immunity against the recurrence of cancer. For example, after administering the vaccine composition as described and exemplified herein as a primary vaccination to the patient, a booster may be administered to support and / or maintain protective immunity. In some aspects, the vaccine composition may be administered to the patient once, twice, or more times per month.

[0141] A vaccination schedule, including primary and booster doses, may be continued over a course of several years, for example, to extend the patient's life as much as necessary. In some aspects, the vaccination schedule involves more frequent administration at the start of vaccination therapy and less frequent administration (e.g., boosters) during the time required to maintain protective immunity.

[0142] The vaccine composition may be administered at a lower dose at the start of vaccine therapy and at a higher dose over time. Additionally, the vaccine may be administered at a higher dose at the start of vaccine therapy and at a lower dose over time.

[0143] The number of administrations of the primary vaccine and booster, and the dosage of the antigen administered, may be tailored and / or adjusted to satisfy the specific needs of individual patients, as determined by the attending physician according to any means suitable in the art.

[0144] A vaccine composition according to one aspect of the present invention is a composition that commonly comprises the aforementioned fusion protein or nanostructure, and to avoid complexity in the specification, the description thereof is omitted within the scope of overlap.

[0145] In one embodiment of the present invention, the vaccine composition is for the prevention or treatment of cancer. When the vaccine composition is for the prevention or treatment of cancer, the antigen of the fusion protein or nanostructure, which is the active ingredient, is a tumor-derived antigen.

[0146] In this specification, the term “prevention” refers to any success or indication of success in preventing the recurrence / relapse of cancer in a patient in a state of clinical remission, as measured by any objective or subjective variable, including the results of radiological or physical examinations.

[0147] Since the fusion protein and nanostructure included in the vaccine composition are identical to the fusion protein and nanostructure described above, the description of overlapping parts is omitted to avoid excessive complexity in the specification.

[0148] According to another aspect of the present invention, the present invention provides a method for preventing or treating an infectious disease or cancer, comprising the step of administering the vaccine composition to a subject.

[0149] The subjects of application of the present invention may be any animal, specifically mammals, e.g., humans, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, monkeys, cattle, horses, pigs; and livestock, including birds, e.g., chickens, ducks, turkeys. The most preferred subjects are livestock or humans.

[0150] A method for preventing or treating an infectious disease or cancer according to one aspect of the present invention includes the step of administering the above-described vaccine composition to a subject; therefore, to avoid complexity in the specification, the description thereof is omitted within the scope of overlap.

[0151] The vaccine composition comprising an assembled protein binding module in addition to the BP26 nanostructure and antigen polypeptide of the present invention is expected to be actively utilized for various medical and health function enhancement purposes, as it has a significant effect in effectively generating antibodies in vivo.

[0152] FIG. 1 is a schematic diagram showing a fusion protein configuration including an assembled protein binding module (a first peptite moiety and a second peptite moiety) of the present invention.

[0153] Figure 2 is a schematic diagram showing a BP26-based nanostructure displaying a SARS-CoV-2 RBD epitope.

[0154] Figure 3 is a schematic diagram showing a BP26-based nanostructure displaying various variants of SARS-CoV-2 RBD epitopes.

[0155] Figure 4 shows a schematic diagram of an experiment to confirm antibody production in mice by nanostructures.

[0156] Figure 5 shows the results of measuring the titer of an anti-RBD IgG antibody produced using BP26-RBD nanostructures.

[0157] Figure 6 shows the results of measuring the antiviral activity of anti-RBD IgG antibodies obtained after prime immunotherapy (D+14) with BP26-RBD nanostructures using CHO cells.

[0158] Figure 7 shows the results of measuring the antiviral activity of anti-RBD IgG antibodies obtained after boosting immunotherapy with BP26-RBD nanostructures (D+35) using CHO cells.

[0159] Figure 8 shows a schematic diagram of an experiment to measure the antiviral activity of a neutralizing antibody prepared using BP26-RBD nanostructures.

[0160] Figure 9 shows the results of measuring the antiviral activity of SARS-CoV-2 neutralizing antibodies produced using BP26-RBD nanostructures.

[0161] Figure 10 shows the immunization schedule of K18-hACE2 mice to evaluate the protective effect of the BP26-RBD nano vaccine against the SARS-CoV-2 virus.

[0162] Figure 11 shows the results of confirming the stability and administration effect of the BP26-RBD nano vaccine in mice by body weight and survival rate.

[0163] To prepare a recombinant protein comprising a BP26 nanostructure, an antigen polypeptide, and an assembled protein binding module, a first peptide moiety bound to the BP26 nanostructure and a second peptide moiety bound to the antigen polypeptide were prepared. A linker was additionally connected between the antigen polypeptide and the second peptide moiety. In the system of the present invention, since the BP26 nanostructure has the characteristic of being capable of self-assembly as an octamer, a single BP26-antigen polypeptide conjugate can be assembled into a hexadecameric hollow barrel structure and can transport multiple (up to 16) antigen polypeptides. This means that a single BP26-antigen polypeptide conjugate can simultaneously transport antigen polypeptides of various variants.

[0164] The present invention will be described in more detail below through examples. These examples are intended solely to explain the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the invention.

[0165] Examples

[0166] In this invention, in vivo data were expressed as the standard error of the mean (± SEM), and other data as the standard deviation (± SD). Each group was compared using one-way analysis of variance (ANOVA) with Tukey multiple comparison tests or two-sided Student's t-tests applied using GraphPad Prism 10 (GraphPad Software). Statistical significance was defined as P < 0.05.

[0167] Preparation Example: Animals, Cells, and Viruses

[0168] Female BALB / c mice (5 weeks old) were purchased from Orient Bio (Seongnam, Korea), and female K18-hACE2 mice (B6.Cg-Tg(K18-ACE2)2Prlmn / J; 6 weeks old) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Mice were housed in groups of 3–5 in standard ventilated cages with soft bedding in a pathogen-free environment with a constant temperature of 23°C and indoor relative humidity of 45–53%, and were provided with free access to standard rodent food and water. After acclimatization for one week, mice weighing 16–18 g were randomly divided into several groups, and all surgical procedures were performed under isoflurane anesthesia to minimize pain and stress. The animal care protocols and experimental procedures were approved by the Animal Care and Use Committee of the Korea Advanced Institute of Science and Technology (KAIST) (Approval No.: KA2021-009). Rhesus monkey kidney cells (Vero 81) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Vero cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; HyClone, CO, USA) supplemented with 10% fetal bovine serum (FBS; Life Technologies, Carlsbad, CA, USA), 100 U / mL penicillin G (Life Technologies), and 100 µg / mL streptomycin (Life Technologies) in a humid environment at 37°C containing 5% CO2. Wild-type SARS-CoV-2 (hCoV-19 / Korea / KCDC06 / 2020) was provided by the Korea Centers for Disease Control and Prevention.

[0169] Example 1: Cloning, expression, and purification of a recombinant protein comprising a BP26 nanostructure, an antigen polypeptide, and an assembled protein binding module

[0170] To prepare a recombinant protein comprising a BP26 nanostructure, an antigen polypeptide, and an assembled protein binding module, a first peptide moiety bound to the BP26 nanostructure and a second peptide moiety bound to the antigen polypeptide were prepared. A linker was additionally connected between the antigen polypeptide and the second peptide moiety (Fig. 1). In the system of the present invention, since the BP26 nanostructure has the characteristic of being capable of self-assembly as an octamer, a single BP26-antigen polypeptide conjugate can be assembled into a hexadecameric hollow barrel structure and can transport multiple (up to 16) antigen polypeptides (Fig. 2). This means that a single BP26-antigen polypeptide conjugate can simultaneously transport antigen polypeptides of various variants (Fig. 3). In the present invention, the receptor binding domain (RBD) of the SARS-Cov2 virus was used as an example of an antigen polypeptide, and the specific sequences used are as shown in Table 3 below.

[0171] Sequence Number Sequence BP261QENQMTTQPARIAVTGEGMMTASPDMAILNLSVLRQAKTAREAMTANNEAMTKVLDAMKKAGIEDRDLQTGGIDIQPIYVYPDDKNNLKEPTITGYSVSTSLTVRVRELANVGKILDESVTLGVNQGGDLNLVNDNPSAVINEARKRAVANAIAKAKTLADAAGVGLGRVVEISELSRPPMPMPIARGQFRTMLAAAPDNSVPIAAGENSYNVSVNVVFEIK BP26 with added His tag and TEV cleavage site (used for cloning Sequence) 2MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSENLYFQGSQENQMTTQPARIAVTGEGMMTASPDMAILNLSVLRQAKTAREAMTANNEAMTKVLDAMKKAGIEDRDLQTGGIDIQPIYVYPDDKNNLK EPTITGYSVSTSLTVRVRELANVGKILDESVTLGVNQGGDLNLVNDNPSAVINEARKRAVANAIAKAKTLADAAGVGLGRVVEISELSRPPMPMPIARGQFRTMLAAAPDNSVPIAAGENSYNVSVNVVFEIKantigen polypeptide3RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF1st peptide moiety4AMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI2nd peptide moiety5AHIVMVDAYKPTKlinker6GSGSGGTEV protease cleavage Sequence 7 ENLYFQG

[0172] Sequences encoding a first peptid moiety (hereinafter referred to as the first module) binding to the BP26 nanostructure and a second peptid moiety (hereinafter referred to as the second module) binding to the antigen polypeptide were synthesized at Genscript (Piscataway, NJ, USA) and inserted into an expression vector. For the expression of the first module protein, the first peptid moiety sequence was genetically linked to the C-terminus of the wild-type BP26 sequence. This sequence was codon-optimized for maximum expression in an E. coli system and inserted into the pET-28a(+)-TEV vector using NdeI and XhoI restriction enzyme sites at the N-terminus and C-terminus of the sequence. The expression vector containing the first module sequence was transformed into BL21(DE3) RIPL E. coli, and the cells were cultured in LB medium containing kanamycin and chloramphenicol for selection. The recombinant protein was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), expressed at 18°C ​​for 16–18 hours, and purified using Ni-NTA affinity chromatography (Qiagen, Hilden, Germany). The protein was further purified using Superdex 200 26 / 60 size exclusion chromatography (GE Healthcare, IL, USA). The expression and purification of the first module recombinant protein were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). For the expression of the second module protein, the second peptid moiety sequence was genetically linked to the C-terminus of the SARS-Cov2 virus RBD sequence. A flexible linker was positioned between the RBD and the second peptid moiety, and a gp67 sequence was added to the N-terminus to promote protein secretion after expression in insect cells. In addition, to remove the gp67 sequence and His tag during the purification process, a TEV protease cleavage sequence was inserted in front of the RBD sequence and the C-terminal 6xHis tag.The sequence was codon-optimized for maximum expression in a Spodoptera frugiperda (Sf9) cell system and inserted into the pFastBac1 vector using BamHI and XhoI restriction enzyme sites at the N-terminus and C-terminus of the sequence. Baculoviruses were generated by transfecting Sf9 cells with an expression vector containing the second module sequence. Sf9 cells were infected with baculoviruses containing the second module sequence, and cells were harvested 48 hours after infection. After cell lysis, the second module protein was purified using Ni-NTA affinity chromatography (Qiagen) and further purified using Superdex 200 26 / 60 size exclusion chromatography (GE Healthcare). The expression and purification of the second module recombinant protein were confirmed by SDS-PAGE.

[0173] Example 2: Production of BP26 nanostructure-antigen polypeptide (BP26-RBD) protein

[0174] The BP26 nanostructure-antigen polypeptide (BP26-RBD) complex was formed by simply mixing the recombinant protein of the first module and the recombinant protein of the second module. To determine the optimal complex formation ratio, the recombinant protein of the first module and the recombinant protein of the second module were mixed at different molar ratios of 2:1, 1:1, 1:2, 1:3, and 1:4 so that the minimum concentration of each protein was 8 pmol. After confirming that 1:2 was the optimal ratio, BP26-RBD was prepared at this ratio. To remove unbound proteins that did not form the BP26-RBD complex, the mixture was further purified using Superdex 200 26 / 60 size exclusion chromatography (GE Healthcare), and the final BP26-RBD product was verified by SDS-PAGE to confirm expression and purification. Endotoxins in the protein solution were removed using a high-volume endotoxin removal spin column (Thermo Fisher Scientific, Waltham, MA, USA), and final endotoxin levels were measured using a colorimetric LAL endotoxin assay kit (GenScript).

[0175] Example 3: Immunization and Measurement of RBD-Specific Antibody Production

[0176] 6-week-old female BALB / c mice were immunized with the prime-boost regimen at 3-week intervals. The mice were divided into four groups and immunized by subcutaneously injecting the vaccines in Table 4 into both soles (Fig. 4).

[0177] Separate administration of vaccine group 1 PBS buffer vehicle group 2 BP26-WT + 2nd module recombinant protein + Alum (simple mixture) group 3 BP26-RBD group 4 BP26-RBD + Alum per mouse, BP26-WT: 9.4 μg; 2nd module recombinant protein: 10.8 μg; BP26-RBD: 25.1 μg.

[0178] The dosage of each modality was adjusted based on an RBD dosage of 10 µg per injection for the RBD administration group. For modalities containing aluminum adjuvants, the commercially available aluminum hydroxide adjuvant Rehydragel HP (Reheis Chemicals, Berkeley Heights, NJ, USA) was used. The antigen solution was mixed with an equal volume of 30% aluminum hydroxide solution and formulated by incubating at room temperature (RT) for 1 hour.

[0179] RBD-specific antibody production was evaluated in mice immunized as described above. Blood was collected from the posterior orbit at three time points: -1 day (the day before the first immunization), 14 days, and 35 days (2 weeks after each immunization). Serum was collected from the blood by centrifugation using a serum separation tube (BD, NJ, USA), and RBD-specific antibody titers were evaluated by enzyme-linked immunosorbent assay (ELISA). Recombinant human coronavirus SARS-CoV-2 spike glycoprotein RBD antigen (Abcam, Cambridge, UK) was coated onto a highly conjugated 96-well plate (Corning, NY, USA) at a concentration of 1 µg / mL and incubated overnight at 4°C. The plates were washed three times with phosphate-buffered saline containing 0.05% Tween 20 (PBST) and then blocked with PBST containing 5% bovine serum albumin (BSA) at room temperature for 1 hour. After washing the plate three times with BST, samples serially diluted in PBS containing 5% BSA were added to the plate and incubated at room temperature for 2 hours. Subsequently, the plate was washed three times again with PBST and incubated at room temperature for 2 hours with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L) antibody (1:10,000 dilution; Thermo Fisher Scientific). After washing the plate and completely removing the liquid, tetramethylbenzidine (TMB) substrate solution (R&D Systems, MN, USA) was added to proceed with development and incubated at room temperature for 20 minutes. Then, 2N sulfuric acid was added to stop the colorimetric reaction. The optical density of each well was measured at 450 nm using a microplate reader (Fig. 4).

[0180] As a result of the test, immunization using the BP26-RBD nanostructure induced the production of anti-RBD IgG antibodies, and antibody titers increased more than 100-fold after multi-boosting immunization. In addition, it was found that administering the fusion protein (BP26-RBD), which includes the BP26 nanostructure, RBD, and assembled protein binding module of the present invention, together with Alum resulted in a higher immune effect; however, the groups administered the fusion protein (BP26-RBD) of the present invention (Groups 3 and 4) all showed higher anti-M2e IgG titers than the group administered a simple mixture of BP26-WT and the second module recombinant protein (Group 2). This implies that using the second module, in which RBD is combined with the second peptide moiety, instead of RBD, can significantly improve efficiency (Fig. 5).

[0181] In addition, to measure the antiviral activity of the above anti-RBD IgG antibody, CHO cells were placed in a 96-well plate at a rate of 3 × 10⁶ per well. 4 After dispensing at a cell density of 10, the cells were infected with SARS-CoV-2 with an infection multiplicity (MOI) of 0.01. As a result of pre-incubation at 37°C for 1 hour with serum series dilutions of 1:10, 1:100, and 1:1,000, similar to the results of anti-RBD IgG antibody production, the antiviral activity of the antibodies was also higher in the group administered with BP26-RBD and Alum (Group 4) than in the group administered with only BP26-RBD without Alum (Group 3), and it was found that the antiviral activity of the antibodies was more significantly enhanced when the second module, in which RBD and the second peptide moiety were combined, was used instead of RBD (Group 2) (Group 4) (Figs. 6 and 7).

[0182] Example 4: Measurement of Antiviral Activity of Neutralizing Antibodies

[0183] Serum was collected from the immunized mice prepared in Example 3 above, and the level of neutralizing antibody production was investigated. Vero 81 cells were placed in a 96-well plate at a rate of 3 × 10⁶ per well.4 After seeding at a cell density, the cells were infected with SARS-CoV-2 with an infection multiplicity (MOI) of 0.01. They were pre-incubated at 37°C for 1 hour with serum series dilutions of 1:10, 1:100, and 1:1,000. At 24 hours post-infection, virus-infected cells were fixed and placed in a cold acetone-methanol solution (1:3 ratio) and incubated at room temperature for 10 minutes to increase permeability. Viral nucleoproteins (NP) were labeled with rabbit anti-NP antibody (GeneTex, Irvine, CA, USA) and Alexa 488-conjugated goat anti-rabbit IgG (Invitrogen, CA, USA), and nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; Invitrogen) (Fig. 8).

[0184] As a result of the test, similar to the anti-RBD IgG antibody production results mentioned above, the antiviral activity of the neutralizing antibody also demonstrated an antiviral effect up to the highest dilution ratio in the group administered BP26-RBD and Alum together (Group 4). The group administered only BP26-RBD (Group 3) had an average of 10 -2 While it maintained antiviral activity up to 1 / 1 dilution, the BP26-RBD / Alum group (Group 4) was 10 -3 Most antiviral activity was maintained even upon dilution. This implies that a more significant immune-boosting effect can be expected when the vaccine composition using the fusion protein of the present invention is administered together with Alum (Fig. 9).

[0185] Example 5: Evaluation of efficacy in preventing viral infection in vivo

[0186] 6-week-old female K18-hACE2 mice were immunized with the Prime-Boost regimen at 3-week intervals, and the mice were divided into four groups and immunized by subcutaneously injecting the vaccine of Table 5 into both soles of the feet.

[0187] Separate Dosage Vaccine Group 1 PBS Buffer Vehicle (without virus) Group 2 Control (SARS-CoV-2 only) Group 3 BP26-RBD 10 µg Group 4 BP26-RBD 5 µg + Alum Group 5 BP26-RBD 10 µg + Alum Group 6 BP26-RBD 20 µg + Alum

[0188] The antigen solution was mixed with an equal volume of 30% aluminum hydroxide solution and formulated by incubating at room temperature (RT) for 1 hour. Two weeks after final immunization, SARS-CoV-2 (2 × 10⁶ per mouse in 50 µl PBS) was administered via the nasal cavity of mice. 4 Mice were infected with plaque-forming units. Body weight and mortality were monitored daily for 14 days after infection, and mice with a 25% decrease in body weight were humanely euthanized and included in the mortality calculation (Fig. 10).

[0189] As a result of the test, the group infected only with the SARS-CoV-2 virus (Group 2) or the group administered only BP26-RBD without Alum after viral infection (Group 3) showed significant weight loss about one week after infection, whereas the groups administered Alum along with BP26-RBD (Groups 4 / 5 / 6) all showed no notable weight loss. In addition, there was no change in survival rate in either the non-viral control group (Group 1) or the groups administered Alum along with BP26-RBD (Groups 4 / 5 / 6). The above results indicate that administering BP26-RBD and Alum together allows for maintaining a health status comparable to that of the non-viral control group (Fig. 11).

[0190] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0191] The vaccine composition comprising an assembled protein binding module in addition to the BP26 nanostructure and antigen polypeptide of the present invention is expected to be actively utilized for various medical and health function enhancement purposes, as it has a significant effect in effectively generating antibodies in vivo.

Claims

1. A fusion protein comprising a BP26 nanostructure, an antigen polypeptide, and an assembled protein binding module.

2. In Paragraph 1, The above BP26 nanostructure is a fusion protein comprising the amino acid sequence of SEQ ID NO.

1.

3. In Paragraph 1, The above-mentioned assembled protein binding module is, A first peptide moiety that binds to the BP26 nanostructure; and, A fusion protein that is a set consisting of a second peptide moiety that binds to an antigen polypeptide.

4. In Paragraph 3, A fusion protein in which the first peptid moiety and the second peptid moiety form an isopeptide bond.

5. In Paragraph 3, The above-mentioned assembled protein binding module set is a fusion protein selected from the group consisting of SnoopTag and SnoopCatcher set, SnoopTagJr and SnoopCatcher set, SnoopTagJr and DogTag set, RrgATag and RrgACatcher set, RrgATag2 and RrgACatcher set, DogTag and RrgACatcher set, SpyTag and SpyCatcher set, IsopepTag and Pilin-C set, IsopepTag-N and Pilin-C set, IsopepTag and Pilin-N set, IsopepTag-N and Pilin-N set, and PsCsTag and PsCsCatcher set.

6. In Paragraph 3, The first peptite moiety above comprises the amino acid sequence of SEQ ID NO. 4, and A fusion protein wherein the second peptite moiety comprises the amino acid sequence of SEQ ID NO.

5.

7. In Paragraph 3, A fusion protein in which a linker is additionally connected between the antigen polypeptide and the second peptide moiety.

8. In Paragraph 7, The above linker is a fusion protein comprising the amino acid sequence of SEQ ID NO.

6.

9. In Paragraph 1, The above antigen is a fusion protein that is an antigen derived from a pathogen or an antigen derived from a tumor.

10. In Paragraph 9, The above pathogen is a fusion protein selected from the group consisting of viruses, bacteria, rickettsia, fungi, and protozoa.

11. A nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein of any one of claims 1 to 10.

12. A recombinant vector comprising the nucleic acid molecule of claim 11.

13. A transformant comprising the recombinant vector of claim 12.

14. A nanostructure comprising the fusion protein of claim 1.

15. In Paragraph 14, The above nanostructure is a nanostructure comprising two or more of the fusion proteins of claim 1.

16. In Paragraph 14, The above nanostructure is a nanostructure comprising 8 or 16 of the fusion proteins of claim 1.

17. A vaccine composition comprising the fusion protein of claim 1, the nanostructure of claim 14, or a combination thereof.

18. In Paragraph 17, The above vaccine composition is a vaccine composition for preventing infectious pathogen infections.

19. In Paragraph 17, The above vaccine composition is a vaccine composition for use in cancer vaccines.

20. In Paragraph 17, The above vaccine composition is a vaccine composition that additionally comprises an adjuvant.

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