CN117425667A - Virus vaccine based on virus surface engineering for enhancing immunity - Google Patents

Abstract

The invention relates to an immunity-enhanced virus vaccine based on virus surface engineering. The connecting peptide according to one aspect has a property of being attachable to a virus, and as a linker, it can effectively bind an immune enhancing substance that activates the immune system to the surface of the virus, thereby enhancing the immunogenicity of the vaccine. By introducing the connecting peptide into a viral surface engineering technique, an immunopotentiating substance can be attached to the viral surface, so that it can be used as an immunopotentiating vaccine platform.

Description

Virus vaccine based on virus surface engineering for enhancing immunity

Technical Field

The invention relates to an immunity-enhanced virus vaccine based on virus surface engineering. The present application claims priority from korean patent application No. 10-2021-0042027, filed on 3 months of 2021, and from korean patent application No. 10-2021-0143999, filed on 26 months of 2021, the contents of which are incorporated herein by reference in their entireties.

Background

Due to the increasing travel of the country, endemic diseases in tropical-subtropical regions are increasingly flowing into the country, and in particular, hundreds of millions of patients are present worldwide due to dengue or novel coronavirus and other infectious diseases. Not only for human beings, but also for animals, the economic losses caused by domestic diseases such as foot-and-mouth disease, african swine fever and the like and foreign input infectious diseases are serious, and the adverse effects on national economy are caused. In order to prevent these viral diseases, development of vaccines is critical, and since it requires a lot of time and expense, it is important to develop vaccine preparation platforms with high efficacy more efficiently.

Generally, there are various types of vaccines, including first-generation vaccines, such as attenuated vaccines, inactivated vaccines, depending on the preparation method; second generation vaccines, e.g., subunit vaccines, toxoid vaccines; and third generation vaccines, such as DNA, RNA, and recombinant viral vaccines. The efficacy of these vaccines varies, as does the efficacy of each type of immune activation. For example, subunit vaccines and viral vaccines, while excellent in safety, require an immunopotentiator or adjuvant (adjuvant) to enhance immunogenicity. An adjuvant is a substance that enhances the immune response to an antigen by inducing a temporary activation of the immune system, and many vaccine companies add adjuvants during vaccine production to improve vaccine efficacy. However, certain adjuvants may cause the vaccine to react too severely, thereby producing side effects such as allergic reactions. Therefore, there is a need for a novel vaccine preparation technology that can maximize the efficacy of a vaccine while minimizing side effects caused by the use of an adjuvant, and that can shorten the vaccine preparation process due to its simplicity. Therefore, a new virus vaccine development platform needs to be constructed to solve the defects of the existing virus vaccine development.

On the other hand, an epitope (epitope) of a virus is usually composed of spike proteins or outwardly protruding protrusions on the surface of the virus, which when bound by antibodies to the surface proteins of the virus can prevent the virus from binding to the receptor of the host cell. The viral surface proteins have the highest immunogenicity and neutralizing capacity, most important in viral infections. For example, in the case of coronaviruses, spike protein (S), membrane protein (M) and envelope protein (E) are protruded as virus structural proteins on the virus surface.

Disclosure of Invention

Technical problem

Against this background, the present inventors prepared novel connecting peptides which have high affinity with other proteins and can be attached to a viral surface, constructed novel vaccine preparation platforms for introducing an immune enhancing substance to a viral surface using the connecting peptides and viral surface engineering techniques, and verified that vaccine compositions prepared using the vaccine platforms have excellent antigenicity, thereby completing the present invention.

One aspect of the present invention provides a connecting peptide consisting of SEQ ID NO:1, and a polypeptide comprising the amino acid sequence of 1.

In another aspect of the invention, there is provided a fusion protein comprising: consists of SEQ ID NO:1, and a linker peptide consisting of the amino acid sequence of 1; and an immunopotentiating substance linked by the C-terminus to the linking peptide.

In yet another aspect of the invention, a vaccine composition is provided comprising an infectious virus-derived antigen and the fusion protein as active ingredients.

In yet another aspect the invention provides a method of preventing or treating an infectious disease, the method comprising administering the vaccine composition to a subject other than a human.

Technical proposal

One aspect of the present invention provides a connecting peptide consisting of SEQ ID NO:1, and a polypeptide comprising the amino acid sequence of 1.

As used herein, the term "peptide" may refer to a linear molecule formed by binding amino acid residues to one another through peptide bonds. The peptides may be prepared according to chemical synthesis methods known in the art, in particular solid phase synthesis techniques or liquid phase synthesis techniques (U.S. patent No. 5,516,891). The present inventors have focused on developing peptides with biologically effective activity, and as a result, identified a peptide consisting of SEQ ID NO:1, and a peptide consisting of the amino acid sequence of 1. Where biologically effective activity may refer to attachment or binding to the surface or at least one region of the virus while retaining the inherent antigenicity of the virus.

As used herein, the term "linker" or "linker peptide" refers to a peptide that links separate polypeptide regions, preferably, a peptide that can directly or indirectly link a viral surface protein and an immune enhancing substance.

The linker peptide may consist of 20 to 30 amino acid sequences, and may also comprise a sequence consisting of SEQ ID NO:1, preferably can be represented by the amino acid sequence set forth in SEQ ID NO:1, and a polypeptide having the amino acid sequence shown in 1. In addition, in addition to the sequence represented by the SEQ ID NO:1, but is not limited to an amino acid sequence having at least 80%, specifically at least 90%, more specifically at least 95%, still more specifically at least 98%, most specifically at least 99% homology with said sequence, provided that it has substantially the same or corresponding efficacy as said protein. Furthermore, as long as the amino acid sequence has such homology, amino acid sequences in which some sequences are deleted, modified, substituted or added are also included in the scope of the present invention, as will be apparent to those skilled in the art.

As used herein, the term "homology" refers to the degree of similarity in the base sequence encoding the protein or the amino acid sequence constituting the protein, and if the homology is sufficiently high, the expression product of the corresponding gene and the protein may have the same or similar activity. The homology may be expressed in terms of a percentage according to the degree of matching with a given amino acid sequence or base sequence, and may be confirmed, for example, by using standard software for calculating parameters (parameter) such as score, identity (identity) and similarity (similarity), specifically using BLAST 2.0, or by comparing sequences by Southern hybridization experiments under defined stringent conditions (stringent condition), which are within the skill of the art, and which may be determined by methods well known to those skilled in the art (e.g., j. Sambrook et al, molecular Cloning, A Laboratory Manual,2nd Edition,Cold Spring Harbor Laboratory press,Cold Spring Harbor,New York,1989;F.M.Ausubel et al, current Protocols in Molecular Biology, john Wiley & Sons, inc., new York).

The connecting peptide has a high affinity with other proteins, in particular, since it can be attached to a viral surface, a vaccine composition having improved immunogenicity over existing viral vaccines can be developed by connecting an immune enhancing substance to viral surface proteins. For example, the linker peptide may be used to link or bind an immunopotentiator to a virus, virus-derived subunit or antigen, virus-like particle. More specifically, the connecting peptide has the ability to bind to virus-derived antigens while effectively retaining antigenicity from the antigens themselves, thereby contributing to improved efficacy as a vaccine composition.

In another aspect of the invention, there is provided a fusion protein comprising: consists of SEQ ID NO:1, and a linker peptide consisting of the amino acid sequence of 1; and an immunopotentiating substance linked by the C-terminus to the linking peptide.

The same parts as described above apply to the fusion protein.

As used herein, the term "immunopotentiator" or "adjuvant" is a substance that can assist an immunogen in developing an immune response, which can function through a variety of mechanisms, including one or more of the following: extending the biological or immunological half-life of the antigen; improving the delivery of antigen to the antigen presentation sequence; improving antigen processing and presentation by antigen presenting cells; and induction of the production of immunomodulatory cytokines.

The immunopotentiator is not particularly limited, but the following may be used alone, or at least two of them may be used: aluminum hydroxide, aluminum phosphate or other aluminum salts, calcium phosphate, DNA CpG motifs, monophosphoryl lipid A, cholera toxin, E.coli heat-inactivated toxin, pertussis toxin, muramyl dipeptide, freund's incomplete adjuvant, MF59, SAF, immunostimulatory complexes, liposomes, biodegradable microspheres, saponins, nonionic block copolymers, muramyl peptide analogues, polyphosphazenes, synthetic polynucleotides, fc regions of antibodies, flagellin (flagellin), IFN-gamma, interleukin-2 (interleukin-2, IL-2) or interleukin-12 (interleukin-12, IL-12), preferably at least one selected from the group consisting of Fc regions of antibodies, flagellin (flagellin) and interleukin-2 (interleukin-12, IL-12) may be used.

The N-terminus of the connecting peptide in the fusion protein can be combined with the virus surface protein, and the C-terminus of the connecting peptide can be combined with the immune enhancing substance. Preferably, the connecting peptides may be arranged in the order of (viral surface protein) - (connecting peptide) - (immunopotentiator), whereby the immunogenicity of the virus may be enhanced.

According to an embodiment, the increased immunogenicity of a vaccine composition according to an aspect of the present invention, in which an antigen peptide that induces a vaccine response against a variety of viruses such as PEDV, PRRSV, SARS-CoV-2, and an Fc-derived protein of IgG are used as an immunopotentiator for effectively inducing antibodies forming the antigen peptide, was confirmed. Thus, the connecting peptide can be used in compositions and methods for preventing or treating infectious diseases caused by viral infection, and the like.

In another aspect the invention provides a polynucleotide encoding said fusion protein, or a recombinant vector comprising said polynucleotide.

As used herein, the term "polynucleotide" as a nucleotide-linked polymeric material refers to DNA encoding genetic information.

In the present invention, in addition to the sequences encoded by SEQ ID NO:1, a base sequence constituting a polynucleotide encoding the connecting peptide further includes, but is not limited to, a base sequence having at least 80%, specifically at least 90%, more specifically at least 95%, still more specifically at least 98%, most specifically at least 99% homology with the sequence, as long as the base sequence constituting a polynucleotide encoding a protein having substantially the same or corresponding efficacy as the respective proteins.

In addition, the polynucleotide encoding the connecting peptide may make various modifications to the coding region within a range that does not alter the amino acid sequence of the protein expressed from the coding region, considering codons preferred in an organism in which the protein is to be expressed due to codon degeneracy (degeneracy). Thus, it may include, but is not limited to, polynucleotides as long as they are nucleotide sequences encoding the respective proteins. In addition, probes that can be prepared according to known sequences, for example, so long as they hybridize under stringent conditions to a sequence complementary to all or part of the polynucleotide sequence to encode a protein having the same activity as the protein, can be included, but are not limited.

By "stringent conditions" is meant conditions that enable specific hybridization between polynucleotides. These conditions are described in detail in the literature (e.g., j. Sambrook et al, supra). For example, the conditions may include: hybridization is performed between genes having higher homology, i.e., genes having at least 40%, specifically at least 90%, more specifically at least 95%, still more specifically at least 97%, most specifically at least 99% homology, while no hybridization is performed between genes having lower homology in comparison; or Southern hybridization, i.e., washed once, specifically twice or three times, at a salt concentration and temperature corresponding to 60℃1 XSSC and 0.1% SDS, specifically 60℃0.1 XSSC and 0.1% SDS, more specifically 68℃0.1 XSSC and 0.1% SDS.

Although there may be mismatches (mismatches) between bases depending on the stringency of hybridization, hybridization requires that the two polynucleotides have complementary sequences. The term "complementary" is used to describe the relationship between nucleotide bases that are hybridizable to each other. For example, with respect to DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Thus, the present description may include not only substantially similar polynucleotides, but also isolated polynucleotide fragments that are complementary to the entire sequence.

Specifically, polynucleotides having homology can be detected using hybridization conditions including a hybridization step at a Tm value of 55℃and the above conditions. In addition, the Tm value may be 60 ℃, 63 ℃ or 65 ℃, but is not limited thereto, and may be appropriately controlled by one of ordinary skill in the art according to the purpose. The appropriate stringency for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, and variables are well known in the art.

As used herein, the term "vector" as a vector that can express a target protein in an appropriate host cell refers to a genetic construct containing a regulatory factor operably linked to express a gene insert. The vector according to an embodiment may include expression regulatory factors such as a promoter, operator, start codon, stop codon, polyadenylation signal and/or enhancer, and the promoter of the vector may be constitutive or inducible. In addition, the vector may be an expression vector that can stably express the fusion protein in a host cell. As the expression vector, a conventional vector used in the art for expressing an exogenous protein in a plant, animal or microorganism can be used. The recombinant vectors shown may be constructed by various methods known in the art. For example, the vector may include a selectable marker for selecting a host cell containing the vector, and if a vector is replicable, an origin of replication.

The vector includes a promoter operable in an animal cell, such as a mammalian cell. According to one embodiment, suitable promoters include promoters derived from mammalian viruses and promoters derived from the genome of mammalian cells, and may include, for example, the Cytomegalovirus (CMV) promoter, the U6 promoter and the H1 promoter, the murine leukemia virus (Murine Leukemia Virus, MLV) long terminal repeat (Long terminal repeat, LTR) promoter, the adenovirus early promoter, the adenovirus late promoter, the vaccine virus 7.5K promoter, the SV40 promoter, the tk promoter of HSV, the RSV promoter, the EF1 a promoter, the metallothionein promoter, the β -actin promoter, the promoter of the human IL-2 gene, the promoter of the human IFN gene, the promoter of the human IL-4 gene, the promoter of the human lymphotoxin gene, the promoter of the human GM-CSF gene, the human phosphoglycerate kinase (PGK) promoter, the mouse phosphoglycerate kinase (PGK) promoter and the Survivin (Survivin) promoter.

In addition, in the vector, the polynucleotide sequence encoding the above fusion protein is operably linked to a promoter. As used herein, the term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcription regulatory factor binding sites) and another nucleic acid sequence whereby the control sequence controls transcription and/or translation of the other nucleic acid sequence.

In another aspect, the invention provides a host cell transformed with the recombinant vector.

As used herein, the term "transformation" is a molecular biological technique in which a DNA strand or plasmid containing a foreign gene of a different kind from the original cell is infiltrated between cells and combined with the DNA of the original cell, thereby changing the genetic characteristics of the cell. For the purposes of the present invention, transformation means that the coding (encoding) comprises the sequence represented by said SEQ ID NO:1 and an immunopotentiating substance linked to the C-terminus of the amino acid sequence, into a host cell to produce it.

The host cell may preferably be any one selected from the group consisting of microorganisms such as bacteria (e.coli) or Yeast (Yeast), CHO cells, F2N cells, and HEK293 cells, but is not limited thereto.

Another aspect of the invention provides a vaccine composition comprising: infectious virus-derived antigen; and a fusion protein comprising a polypeptide consisting of SEQ ID NO:1 and an immunopotentiator attached to the C-terminus of said linker peptide.

The same parts as described above apply to the vaccine composition.

As used herein, the term "vaccine" as a biological agent containing an antigen that causes an organism to develop immunity, refers to an immunogen or antigenic material that is used to develop immunity in vivo by injection or oral administration to a human or animal to prevent infectious disease. In vivo immunity is largely divided into active immunity, which means that in vivo immunity is automatically obtained after infection with a pathogen, and passive immunity, which means that in vivo immunity is obtained by external injection of a vaccine. Active immunization has the characteristic of long-term and continuous immunization of antibodies related to immunization, while passive immunization generated by a vaccine can immediately play a role in treating infectious diseases, but has the disadvantage of poor persistence. The vaccine may be used interchangeably with the term "immunogenic composition", e.g., it may be an immunogenic composition against porcine epidemic diarrhea virus or porcine reproductive and respiratory syndrome virus infection, but is not limited thereto.

As used herein, the term "immunogen" or "antigenic material" may be any one selected from the group consisting of peptides derived from said virus, polypeptides, lactic acid bacteria expressing said polypeptides, proteins, lactic acid bacteria expressing said proteins, oligonucleotides, polynucleotides and recombinant viruses. As specific examples, the antigenic material may be in the form of an inactivated whole or part of a viral preparation, or in the form of an antigenic molecule obtained by conventional protein purification, genetic engineering techniques or chemical synthesis.

In a specific embodiment, the virus is not particularly limited, but may be an RNA-type virus or a DNA-type virus.

The RNA virus is a general term for viruses having RNA as a gene, and includes: viruses having (+) strand RNA, complementary strand (-) strand RNA, and double-stranded RNA as mRNA as genes in viral particles; viruses with only one RNA molecule (coronaviruses, paramyxoviruses); viruses (retroviruses) having two identical types of RNA molecules; and viruses (influenza viruses) having eight different RNA molecules as genes, etc. In general, RNA synthetase (DNA synthetase in the case of retrovirus) exists using RNA as a template.

DNA viruses are classified into circular DNA viruses and linear DNA viruses according to the shape of genes. The linear DNA viruses include parvoviruses having single-stranded linear DNA in viral particles, and adenoviruses, herpesviruses, poxviruses, and the like having double-stranded linear DNA. Most have specific repeats at the ends of the genome and, due to differences in genome structure or size, exhibit inherent modes of infection and proliferation, respectively. DNA viruses that use circular DNA molecules as their genome are largely divided into two viral families. That is, viruses of the family Paramyviridae (polyomaviruses, SV40, papillomaviruses, etc.) whose genomes are double-closed-loop DNA molecules, and viruses of the family Hepadnaviridae (hepadnavidae) whose genomes are double-loop DNA molecules comprising single-stranded portions (hepatitis B virus, woodchuck) hepatitis virus, etc.).

In the present specification, viruses to which the connecting peptide can bind to the surface protein are not particularly limited, and may include RNA-type viruses or DNA-type viruses as described above.

In a specific embodiment, the infectious virus-derived antigen may include, but is not limited to, antigens derived from porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus), porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus), dengue virus (Dengue virus), japanese encephalitis virus (Japanese encephalitis virus), zika virus (Zika virus), ebola virus (Ebola virus), rotavirus (Rotavirus), dengue virus (Dengue virus), west Nile virus (West Nile virus), yellow fever virus (Yellow fever virus), adenovirus (adenovus), BK virus (BK virus), smallpox virus (Smallpox virus), severe fever with thrombocytopenia syndrome virus (Severe fever with thrombocytopenia syndrome virus), herpes simplex virus (Herpes simplex virus), epstein-Barr virus (Epstein-Barr virus), hepatitis a virus (Hepatitis A virus), hepatitis b virus (Hepatitis B virus), hepatitis c virus (Hepatitis C virus), hepatitis delta virus (Hepatitis D virus), hepatitis e virus (Hepatitis E virus), hepatitis c virus (cyto virus), or hantavirus (cyto virus).

In a specific embodiment, the vaccine composition is an attenuated live vaccine (Live attenuated vaccine), an inactivated vaccine (Inactivated vaccine), a Subunit vaccine (subnit vaccinee), or a virus-like particle vaccine (Virus like Particle vaccine).

As used herein, the term "Virus-Like Particle (VLP)" may refer to non-infectious viral subunits with or without viral proteins. For example, the virus-like particle may refer to a recombinant protein having a form similar to that of a virus, which self-assembles (self-assembly) into a form similar to that of an actual virus through binding between virus structural proteins, but during assembly, a virus gene may not be contained inside the virus-like particle. The virus-like particle having such characteristics is very similar to the form of an actual virus, so that it can exhibit high immunogenicity after injection into the body, and since it does not contain viral genes, it can be used as a safety antigen that cannot be reproduced in the body.

The virus-like particles may include viruses, e.g., spike proteins, membrane proteins, envelope proteins, and nucleocapsid proteins of coronaviruses. Wherein, spike protein is a structural protein existing on the surface of virus and consists of rod-shaped protrusions. Binding of the protein to glycoprotein receptors of host cells is known to result in fusion of the cell membrane with the viral outer membrane, and production of neutralizing antibodies. In addition, nucleocapsid proteins are known to exist inside the outer shell and are involved in cellular immune responses.

The vaccine composition may further comprise a pharmaceutically acceptable excipient, diluent or carrier. The "pharmaceutically acceptable excipient, diluent or carrier" may refer to an excipient, diluent or carrier that does not inhibit the biological activity and properties of the injected compound while not stimulating the organism. Wherein "pharmaceutically acceptable" means that the active ingredient does not inhibit its activity without having a degree of toxicity beyond that acceptable for the subject to whom it is applied (prescribed).

Suitable vectors for vaccines are known to those skilled in the art and include, but are not limited to, proteins, sugars, and the like. The carrier may be an aqueous solution, or a non-aqueous solution, suspension or emulsion. As an adjuvant for increasing immunogenicity, a shaped or amorphous organic or inorganic polymer or the like can be used. Immunoadjuvants are known to generally function to promote an immune response by chemical and physical binding to an antigen. As the adjuvant, amorphous aluminum gel, oil emulsion, double oil emulsion, immune sol (Immunosol) and the like can be used. In addition, in order to promote immune response, various plant-derived saponins, levamisole, cpG dinucleotides, RNA, DNA, LPS, various types of cytokines, and the like can be used. The immune composition as described above can be used for a composition for inducing an optimal immune response by combining various adjuvants and additives for promoting an immune response. In addition, the compositions that may be added to the vaccine may include stabilizers, inactivating agents, antibiotics, preserving agents, and the like. Depending on the route of administration of the vaccine, the vaccine antigen may be used in admixture with distilled water, a buffer solution, or the like.

The vaccine compositions may be formulated and used in conventional manner in oral dosage forms, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or unit dose ampoules or multi-dose forms of injection, respectively. When formulating the vaccine composition, it may be prepared by adding diluents or excipients, such as usual fillers, extenders, binders, wetting agents, disintegrants or surfactants.

When the vaccine composition is prepared as a parenteral dosage form, it may be formulated with a suitable carrier in the form of injections, transdermal administration, nasal inhalants and suppositories according to methods known in the art. When formulated as an injection, suitable carriers may include sterile water, ethanol, polyols such as glycerol or propylene glycol, and the like, or mixtures thereof, and isotonic solutions such as ringer's solution, triethanolamine-containing phosphate buffer (phosphate buffered saline, PBS), or sterile water for injection, 5% dextrose, and the like may be preferably used. When formulated into a transdermal administration preparation, it may be formulated into the form of ointments, creams, emulsions, gels, solutions for external use, pastes, liniments, aerosols, etc. For nasal inhalants, suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and carbon dioxide may be used to formulate as aerosol sprays, and when formulated as suppositories, witepsol (witepsol), tween (tween) 61, polyethylene glycols, cocoa butter, lauryl esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters and the like may be used.

The administration route of the vaccine composition may be any conventional route so long as the target tissue can be reached, and in particular, the vaccine composition may be selected from compositions for intramuscular administration, subcutaneous administration, intraperitoneal administration, intravenous administration, oral administration, dermal administration, ocular administration, and brain administration.

The vaccine composition may be administered in a pharmaceutically effective amount, the term "pharmaceutically effective amount" referring to an amount sufficient to treat or prevent a disease and having a reasonable benefit/risk ratio applicable to medical treatment or prevention, and the effective amount level may be determined according to the following factors: the severity of the disease, the activity of the drug, the age, weight, health, sex, sensitivity of the patient to the drug, the time of administration of the composition of the invention used, the route of administration and rate of excretion, the period of treatment, the drug combined with or concurrent with the composition of the invention used, and other factors known in the medical arts. The vaccine composition may be administered alone or in combination with ingredients known to exhibit prophylactic or therapeutic effects on known infectious diseases. It is important to consider the factors in combination that the amount that is capable of achieving the greatest effect in the smallest amount and without side effects is administered.

The person skilled in the art can determine the dosage of the vaccine composition by considering the purpose of use, the degree of addiction to the disease, the age, weight, sex, medical history of the patient or the type of substance used as active ingredient. For example, the vaccine composition of the present invention may be administered at about 0.1ng to about 1000mg/kg, preferably 1ng to about 100mg/kg per adult, and the administration frequency of administration of the composition of the present invention is not particularly limited, but may be administered once daily or may be divided into several administrations. The dosage or frequency of administration does not limit the scope of the invention in any way.

In another aspect the invention provides a method of preventing or treating an infectious disease comprising administering the vaccine composition to a subject. The same parts as described above apply to the method.

As used herein, the term "preventing" refers to any act of inhibiting or delaying the infection of an infectious disease and the onset of the infectious disease by administering the vaccine composition.

As used herein, the term "treatment" refers to any action that ameliorates or benefits the symptoms of a disease that has been caused by an infectious disease infection by administering the vaccine composition.

As used herein, the term "infectious disease" refers to a viral infectious disease, preferably a disease caused by a viral infection, but is not limited thereto.

The subject may include, but is not limited to, mammals, farmed fish, etc., such as cows, horses, sheep, pigs, goats, camels, antelopes, dogs, cats, rats, livestock, humans, etc., that have developed or are likely to develop viral infections and diseases caused by the infections.

As used herein, the term "administering" refers to administering a given substance to a subject by an appropriate method, and the route of administration of the vaccine composition of the present invention may be any conventional route, as long as the target tissue is reached. The administration may include, but is not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary or administration, and intrarectal administration. However, when administered orally, the oral composition is preferably formulated as a coated active agent or to protect it from decomposition in the stomach, as the protein is digested. Alternatively, the pharmaceutical composition may be administered by any means that can deliver the active agent to the target cell.

The vaccine composition may be administered as a sole therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with existing therapeutic agents. And may be administered in a single or multiple applications. Importantly, taking the factors into consideration, the amount that is administered to achieve the greatest effect in the smallest amount and without side effects can be readily determined by one of skill in the art.

Advantageous effects

The connecting peptide according to one aspect has a property of being attachable to a virus, and as a linker, it can effectively bind an immune enhancing substance that activates the immune system to the surface of the virus, thereby enhancing the immunogenicity of the vaccine. The connecting peptide can be introduced into a virus surface engineering technology to attach an immune enhancing substance on the surface of the virus, so that the connecting peptide can be used as an immune enhancing vaccine platform.

Brief description of the drawings

Fig. 1 is a result of confirming expression of a connecting peptide (VSE peptide) according to an aspect by western blotting.

Fig. 2 is a graph showing the results of confirming the adhesion activity of a connecting peptide to a viral surface according to an aspect, wherein a of fig. 2 schematically shows the formation of an antigen-antibody complex and detection thereof for evaluation of the adhesion activity to a viral surface, and B of fig. 2 is a graph showing the results of evaluation of the adhesion activity to a viral surface quantitatively.

FIG. 3 is a result of confirming expression of recombinant proteins (VSE-hFc, VSE-sFc) according to an aspect by western blotting.

Fig. 4 is a graph showing the results of confirming the adhesion activity of a recombinant protein to a viral surface according to an aspect, wherein a of fig. 4 schematically shows the formation of an antigen-antibody complex and detection thereof for evaluation of the adhesion activity to a viral surface, and B of fig. 4 is a graph showing the results of evaluation of the adhesion activity to a viral surface quantitatively.

FIG. 5 is a diagram of a process for preparing a recombinant antigen (PEDV-VSE-sFc) according to an aspect.

FIG. 6 is a result of confirming the level of PEDV-VSE-sFc present in serum of a mouse after intraperitoneal administration of PEDV-VSE-sFc according to an aspect to the mouse.

FIG. 7 is a graph of the results of confirming the level of neutralizing antibodies present in the serum of mice after intraperitoneally administering PEDV-VSE-sFc according to one aspect to the mice.

FIG. 8 is a diagram of a process for preparing a recombinant antigen (DENV-VSE-hFc) according to an aspect.

FIG. 9 is a result of confirming the levels of DENV-specific IgG present in serum of a mouse after intraperitoneal administration of DENV-VSE-hFc according to an aspect to the mouse.

FIG. 10 is a result of confirming neutralizing antibody levels present in serum of mice after intraperitoneal administration of DENV-VSE-hFc according to an aspect to the mice.

FIG. 11 is a result of confirming expression of recombinant antigen (PEDV-Fc) according to an aspect by western blotting.

FIG. 12 is a result of confirming the levels of IgG present in serum and colostrum of an animal model after administration of PEDV-Fc according to one aspect to the animal model.

FIG. 13 is a result of confirming the level of neutralizing antibodies present in serum of an animal model after administration of PEDV-Fc according to an aspect to the animal model.

FIG. 14 is a result of confirming expression of recombinant antigen (PRRSV-Fc) according to an aspect by Western blotting.

Fig. 15 is a result of confirming IgG levels present in serum and colostrum of an animal model after administration of PRRSV-Fc according to an aspect to the animal model.

Best mode

Described in more detail below with reference to examples. However, these examples are for descriptive purposes only and the scope of the invention is not limited to these examples.

Example 1: construction of the connecting peptide

A linker peptide (VSE peptide) having an effective binding capacity to a viral surface or a virus-derived antigen is derived and prepared. The amino acid sequence of the linker peptide and the polynucleotide sequence encoding it are shown in table 1 below.

[ Table 1 ]

In this example, the VSE polynucleotide was cloned into eukaryotic cell expression vector pcdna3.1-Myc-His vector, expressed in CHO cells, and purified using Myc tags to obtain VSE peptide, as shown in fig. 1.

Example 2: determination of attachment Activity against viral surface

In this example, the adhesion activity of VSE peptides to viral surfaces was confirmed by enzyme-linked immunosorbent assay (ELISA). Specifically, after Porcine Epidemic Diarrhea Virus (PEDV), porcine Reproductive and Respiratory Syndrome Virus (PRRSV), dengue virus (DENV), japanese Encephalitis Virus (JEV) or ZIKV virus (ZIKV) were coated on the surface of an immune plate (immunoplate), myc-labeled VSE peptide (VSE-Myc tag) of example 1 was added to induce an attachment/binding reaction, respectively. Then, after induction of the reaction by adding an anti-Myc tag antibody labeled with horseradish peroxidase (Horseradish peroxidase, HRP), the adhesion activity to the virus surface was evaluated by quantitatively detecting the level of HRP (fig. 2A). On the other hand, the negative control group was set as a group that reacted with the disordered peptide (Scramble pepetide), and the positive control group was set as a group that used the serum of mice immunized with each virus.

As a result, as shown in fig. 2B, the detection level of the labeled HRP was very low in the negative control group, while a similar level to that of the positive control group was detected in the group using the VSE peptide according to an aspect. These experimental results demonstrate that VSE peptides according to one aspect have potent adhesion activity to viral surfaces.

Example 3: determination of adhesion Activity of immunopotentiating substances against viral surface

In this example, the adhesion activity of the VSE peptide to the viral surface is used to attach or bind an immunopotentiator to the viral surface. Specifically, a VSE peptide and a human Fc (VSE-hFc) or a porcine (sWine) Fc (VSE-sFc) were cloned into a eukaryotic cell expression vector pcDNA3.1-Myc-His vector and expressed in CHO cells, as shown in FIG. 3, to obtain a recombinant protein VSE-hFc or VSE-sFc containing an immunopotentiating substance. The amino acid sequences of the recombinant proteins (VSE-hFc, VSE-sFc) and the polynucleotide sequences encoding them are shown in tables 2 and 3 below.

[ Table 2 ]

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[ Table 3 ]

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Next, after Porcine Epidemic Diarrhea Virus (PEDV), porcine Reproductive and Respiratory Syndrome Virus (PRRSV), dengue virus (DENV), japanese Encephalitis Virus (JEV) or ZIKV is coated on the surface of an immune plate (immunoplate), respectively, the VSE-hFc or VSE-sFc is added to induce an attachment/binding reaction. Then, after induction of a reaction by adding an anti-IgG antibody labeled with horseradish peroxidase (Horseradish peroxidase, HRP), the adhesion activity to the virus surface was evaluated by quantitatively detecting the level of HRP (a of fig. 4). On the other hand, the negative control group was set as a group using a disorder peptide (Scramble peptide), and the positive control group was set as a group using mouse serum immunized with each virus.

As a result, as shown in B of fig. 4, the detection level of the labeled HRP was very low in the negative control group, while the similar level to that in the positive control group was detected in the group (VSE-hFc, VSE-sFc) using the VSE peptide according to the aspect. These experimental results demonstrate that VSE peptides according to one aspect can help to improve the efficacy of vaccine formulations containing viral antigens by attaching or binding immune enhancing substances to the surface of the viral antigen.

Example 4: confirmation of immune response enhancing Effect by Virus surface engineering

In this example, after preparing a recombinant antigen by attaching/binding VSE-sFc to the surface of a viral antigen, the immune response enhancing effect of the recombinant antigen was confirmed using a mouse model.

4-1、PEDV-VSE-sFc

As shown in FIG. 5, after mixing Porcine Epidemic Diarrhea Virus (PEDV) with VSE-sFc of example 3, an attachment/binding reaction therebetween was induced at room temperature for 2 hours to prepare recombinant antigen (PEDV-VSE-sFc).

Specifically, after 3 intraperitoneal injections of 4-week-old Balb/C mice with PEDV-VSE-sFc for immunization at two weeks intervals, the level of PEDV-specific IgG present in the serum of the mice was confirmed. At the same time, the serum of the immunized mice was subjected to a plaque reduction neutralization test (Plaque reduction neutralization test) to evaluate the level of neutralizing antibodies against PEDV antigen. On the other hand, the group to which PBS was applied was set as a negative control group, and the group to which only PEDV was applied was set as a comparative group.

As a result, as shown in fig. 6 and 7, in the group to which PEDV-VSE-sFc was administered according to an aspect, high levels of IgG and neutralizing antibodies were confirmed in the serum of mice. In particular, the level of neutralizing antibodies in serum of PEDV-VSE-sFc immunized mice was increased by about 4.5 fold compared to the comparative group.

4-2、DENV-VSE-hFc

As shown in FIG. 8, after dengue virus (DENV) was mixed with VSE-hFc of example 3, an attachment/binding reaction therebetween was induced at room temperature for 2 hours to prepare recombinant antigen (DENV-VSE-hFc).

Specifically, levels of DENV-specific IgG present in mouse serum were confirmed 3 times following intraperitoneal injection of 4-week-old Balb/C mice with DENV-VSE-hFc for immunization at two week intervals. At the same time, a plaque reduction neutralization assay (Plaque reduction neutralization test) was performed on the serum of the immunized mice to assess neutralizing antibody levels against DENV antigen. On the other hand, the group to which PBS was applied was set as a negative control group, and the group to which DENV alone was applied was set as a comparative group.

As a result, as shown in fig. 9 and 10, in the group to which DENV-VSE-hFc was administered according to an aspect, high levels of IgG and neutralizing antibodies were confirmed in the serum of mice. In particular, the levels of neutralizing antibodies in serum of DENV-VSE-hFc immunized mice were increased by about 4.5 fold as compared to the comparative group.

Summarizing these experimental results, it was found that the immune response-inducing effect of viral antigens was significantly improved when an immune enhancing substance was attached to the viral surface using the VSE peptide according to an aspect. Thus, the recombinant antigen shows improved efficacy as an active ingredient of a vaccine formulation.

Example 5: confirmation of the immune response-enhancing Effect against virus-derived antigen

In this example, after preparing a recombinant antigen by attaching/binding VSE-sFc to a virus-derived antigen, the immune response enhancing effect of the recombinant antigen was confirmed.

5-1, PEDV-derived spike protein

Recombinant antigens containing the spike protein S1-derived protein of PEDV, which is an antigen that can induce a vaccine response against PEDV, and the Fc-derived protein of IgG, which is an immunopotentiator for enhancing antibody formation, were prepared in the same manner as described in example 4 using the VSE peptide. In addition, it was expressed in CHO cells to obtain PEDV-Fc, which is a recombinant antigen containing an immunopotentiating substance, as shown in fig. 11. The amino acid sequences of the VSE peptide-Fc derived proteins used in the recombinant antigen (PEDV-Fc) are shown in Table 4 below.

[ Table 4 ]

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In addition, the following experiments were performed to confirm the immunogenicity of the immunopotentiated PEDV virus vaccine. The experimental animals were injected intramuscularly with the prepared vaccine (PEDV-Fc) twice, at intervals of two weeks (dose: 100 ul). Two weeks after the second inoculation, serum and colostrum were collected to measure IgG titers, and ELISA and neutralization capacity assays were performed to detect antibody titers in the serum and colostrum. On the other hand, the group to which PBS was applied was set as a control group, and the group to which only PEDV was applied was set as a comparative group.

As a result, as shown in fig. 12 and 13, the recombinant antigen according to an example showed higher levels of IgG titers in the serum and colostrum of the sow as compared to the comparative group, and the serum-neutralizing antibodies also showed the same tendency as described above.

5-2, PRRSV derived GP5 proteins

Recombinant antigens containing GP5 protein of PRRSV, which is an antigen that can induce a vaccine response against PRRSV, and Fc-derived protein of IgG, which is an immunopotentiator for enhancing antibody formation, were prepared in the same manner as described in example 4 using VSE peptide. In addition, it was expressed in CHO cells and Marc145 cells to obtain PRRSV-Fc, which is a recombinant antigen containing an immune enhancing substance, as shown in fig. 14. The amino acid sequences of the VSE peptide-Fc derived proteins used in the recombinant antigens (PRRSV-Fc) are shown in table 5 below.

[ Table 5 ]

In addition, the following experiments were performed to confirm the immunogenicity of the immunopotentiated PRRS virus vaccine. The experimental animals were intramuscular injected twice with the prepared vaccine (PRRSV-Fc) at two week intervals (dose: 100 ul). Two weeks after the second inoculation, serum and colostrum were collected to measure IgG titers, and ELISA and neutralization capacity assays were performed to detect antibody titers in the serum and colostrum. On the other hand, the PBS-applied group was set as a control group, and the PRRSV-only group was set as a comparative group.

As a result, as shown in fig. 15, the recombinant antigen according to an example showed higher levels of IgG titers in both serum and colostrum of sows compared to the comparative group.

The experimental results demonstrate that the vaccine composition according to an embodiment has increased immunogenicity, so that the efficacy of the vaccine can be maximized.

The above description of the present invention is merely for example, and it will be understood by those skilled in the art that the present invention may be easily modified into other specific forms without changing the technical spirit or essential characteristics of the present invention. Accordingly, it should be understood that the embodiments described above are illustrative in all respects, rather than restrictive.

Sequence listing

<110> Cooperation of university of loyal south university (IAC)

<120> surface engineered adjuvanted viral vaccines

<130> PX067908PCT

<150> KR 10-2021-0042027

<151> 2021-03-31

<150> KR 10-2021-0143999

<151> 2021-10-26

<160> 8

<170> PatentIn version 3.2

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His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

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Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

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Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

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Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

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ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 180

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gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 420

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 480

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gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 600

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 660

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Pro Leu Ala Pro Cys Gly Arg Asp Val Ser Asp His Asn Val Ala Leu

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Gly Cys Leu Val Ser Ser Tyr Phe Pro Glu Pro Val Thr Val Thr Trp

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Asn Ser Gly Ala Leu Ser Arg Val Val His Thr Phe Pro Ser Val Leu

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Thr Ala Gln Thr Arg Pro Met Glu Glu Gln Phe Asn Ser Thr Tyr Arg

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Val Val Ser Val Leu Pro Ile Gln His Gln Asp Trp Leu Lys Gly Lys

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Gln Ala Pro Ala Val Val Val Leu Gly Gly Tyr Leu Pro Ser Met Asn

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Ser Ser Ser Trp Tyr Cys Gly Thr Gly Ile Glu Thr Ala Ser Gly Val

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His Gly Ile Phe Leu Ser Tyr Ile Asp Ser Ser Gln Gly Phe Glu Ile

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Gly Ile Ser Gln Glu Pro Phe Asp Pro Ser Gly Tyr Gln Leu Tyr Leu

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His Lys Ala Thr Asn Gly Asn Thr Asn Ala Ile Ala Arg Leu Arg Ile

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Ser Gln Phe Pro Asp Asn Lys Thr Leu Gly Pro Thr Val Asn Asp Val

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Thr Thr Gly Arg Asn Cys Leu Phe Asn Lys Ala Ile Pro Ala Tyr Met

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Arg Asp Gly Lys Asp Ile Val Val Gly Ile Thr Trp Asp Asn Asp Arg

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Val Thr Val Phe Ala Asp Lys Ile Tyr His Phe Tyr Leu Lys Asn Asp

195 200 205

Trp Ser Arg Val Ala Thr Arg Cys Tyr Asn Arg Arg Ser Cys Ala Met

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Gln Tyr Val Tyr Thr Pro Thr Tyr Tyr Met Leu Asn Val Thr Ser Ala

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Gly Glu Asp Gly Ile Tyr Tyr Glu Pro Cys Thr Ala Asn Cys Thr Gly

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Tyr Ala Ala Asn Val Phe Ala Thr Asp Ser Asn Gly His Ile Pro Glu

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Ser Phe Ser Phe Asn Asn Trp Phe Leu Leu Ser Asn Asp Ser Thr Leu

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Leu His Gly Lys Val Val Ser Asn Gln Pro Leu Leu Val Asn Cys Leu

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Leu Ala Ile Pro Lys Ile Tyr Gly Leu Gly Gln Phe Phe Ser Phe Asn

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His Thr Met Asp Gly Val Cys Asn Gly Ala Ala Leu Asp Arg Ala Pro

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Glu Ala Leu Arg Phe Asn Ile Asn Asp Thr Ser Val Ile Leu Ala Glu

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Gly Ser Ile Val Leu His Thr Ala Leu Gly Thr Asn Leu Ser Phe Val

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Cys Ser Asn Ser Ser Asp Pro His Leu Ala Thr Phe Ala Ile Pro Leu

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Gly Ala Thr Glu Val Pro Tyr Tyr Cys Phe Leu Ile Val Asp Thr Tyr

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Asn Ser Thr Val Tyr Lys Phe Leu Ala Val Leu Pro Pro Thr Val Arg

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Glu Ile Val Ile Thr Lys Tyr Gly Asp Val Tyr Val Asn Gly Phe Gly

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Tyr Leu His Leu Gly Leu Leu Asp Ala Val Thr Ile Asn Phe Thr Gly

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His Gly Thr Asp Asp Asp Val Ser Gly Phe Trp Thr Ile Asp Ser Thr

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Asn Phe Val Asp Ala Leu Ile Glu Val Gln Gly Thr Ser Ile Gln Arg

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Ile Leu Tyr Cys Asp Asp Pro Val Ser Gln Leu Lys Cys Ser Gln Val

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Ala Phe Asp Leu Asp Asp Gly Phe Tyr Pro Ile Ser Ser Arg Asn Leu

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Leu Ser His Glu Gln Pro Ile Ser Phe Val Thr Leu Pro Ser Phe Asn

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Asp His Ser Phe Val Asn Ile Thr Val Ser Ala Ser Phe Gly Asp His

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Ser Gly Ala Asn Leu Val Ala Ser Asp Thr Thr Ile Asn Gly Phe Ser

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Ser Phe Cys Val Asp Thr Arg Gln Phe Thr Ile Arg Leu Phe Tyr Asn

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Val Thr Ser Ser Tyr Gly Tyr Val Ser Lys Ser Gln Tyr Ser Asn Cys

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Pro Phe Thr Leu Gln Ser Val Asn Asp Tyr Leu Ser Phe Ser Lys Phe

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Cys Val Ser Thr Ser Leu Leu Ala Ser Ala Cys Thr Ile Asp Leu Phe

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Gly Tyr Pro His Phe Gly Ser Gly Val Lys Phe Thr Ser Leu Tyr Phe

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Gln Phe Thr Glu Gly Glu Leu Ile Thr Gly Thr Pro Lys Pro Leu Glu

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Gly Val Thr Asp Val Ser Phe Met Thr Leu Asp Val Cys Thr Lys Tyr

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Thr Ile Tyr Gly Phe Lys Gly Glu Gly Ile Ile Thr Leu Thr Asn Ser

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Ser Phe Leu Ala Gly Val Tyr Tyr Thr Ser Asp Ser Gly Gln Leu Leu

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Ala Phe Lys Asn Val Thr Ser Gly Ala Val Tyr Ser Val Thr Pro Cys

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Ser Phe Ser Glu Gln Ala Ala Tyr Val Asp Asp Asp Ile Val Gly Val

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Ile Ser Ser Leu Ser Asn Ser Thr Phe Asn Asn Thr Arg Glu Leu Pro

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Gly Phe Phe Tyr His Ser Asn Asp Gly Ser Asn Cys Thr Glu Pro Val

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Leu Val Tyr Ser Asn Ile Gly Val Cys Lys Ser Gly Ser Ile Gly Tyr

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Val Pro Ser Gln Ser Gly Gln Val Lys Ile Ala Pro Thr Val Thr Gly

785 790 795 800

Asn Ile Ser Ile Pro Thr Asn Phe Ser Met Ser Ile Arg Thr Glu Tyr

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Leu Gln Leu Tyr Asn Thr Pro Val Ser Val Asp Cys Ala Thr Tyr Val

820 825 830

Cys Asn Gly Asn Ser Arg Cys Lys Gln Leu Leu Thr Gln Tyr Thr Ala

835 840 845

Ala Cys Lys Thr Ile Glu Ser Ala Leu Gln Leu Ser Ala Arg Leu Glu

850 855 860

Ser Val Glu Val Asn Ser Met Leu Thr Thr Ser Glu Gln Ala Leu Gln

865 870 875 880

Leu Ala Thr Ile Ser Ser Phe Asn Gly Asp Gly Tyr Asn Phe Thr Asn

885 890 895

Val Leu Gly Val Ser Val Tyr Asp Pro Ala Ser Gly Arg Val Val His

900 905 910

Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Val Thr Asn

915 920 925

Gly Leu Gly Thr Val Asp Glu Asp Tyr Lys Arg Cys Ser Asn Gly Arg

930 935 940

Ser Val Ala Asp Leu Val Cys Ala Gln Tyr Tyr Ser Gly Val Met Val

945 950 955 960

Leu Pro Gly Val Val Asp Ala Glu Lys Leu His Met Tyr Ser Ala Ser

965 970 975

Leu Ile Gly Gly Met Val Leu Gly Gly Phe Thr Ala Ala Ala Ala Leu

980 985 990

Pro Phe Ser Tyr Ala Val Gln Ala Arg Leu Asn Tyr Leu Ala Leu Gln

995 1000 1005

Thr Asp Val Leu Gln Arg Asn Gln Gln Leu Leu Ala Glu Ser Phe

1010 1015 1020

Asn Ser Ala Ile Gly Asn Ile Thr Ser Ala Phe Glu Ser Val Lys

1025 1030 1035

Glu Ala Ile Ser Gln Thr Ser Gln Gly Leu Asn Thr Val Ala Arg

1040 1045 1050

Ala Leu Thr Lys Val Gln Glu Val Val Asn Ser Gln Gly Ala Ala

1055 1060 1065

Leu Thr Gln Leu Thr Val Gln Leu Gln His Asn Phe Gln Ala Ile

1070 1075 1080

Ser Ser Ser Ile Asp Asp Ile Tyr Ser Arg Leu Asp Ile Leu Ser

1085 1090 1095

Ala Asp Val Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Ser Ala

1100 1105 1110

Leu Asn Ala Phe Val Ala Gln Thr Leu Thr Lys Tyr Thr Glu Val

1115 1120 1125

Gln Ala Ser Arg Lys Leu Ala Gln Gln Lys Val Asn Glu Cys Val

1130 1135 1140

Lys Ser Gln Ser Gln Arg Tyr Gly Phe Cys Gly Gly Asp Gly Glu

1145 1150 1155

His Ile Phe Ser Leu Val Gln Ala Ala Pro Gln Gly Leu Leu Phe

1160 1165 1170

Leu His Thr Val Leu Val Pro Gly Asp Phe Val Asn Val Ile Ala

1175 1180 1185

Ile Ala Gly Leu Cys Val Asn Gly Asp Ile Ala Leu Thr Leu Arg

1190 1195 1200

Glu Pro Gly Leu Val Leu Phe Thr His Glu Leu Gln Thr His Thr

1205 1210 1215

Ala Thr Glu Tyr Phe Val Ser Ser Arg Arg Met Phe Glu Leu Arg

1220 1225 1230

Lys Pro Thr Val Ser Asp Phe Val Gln Ile Glu Ser Cys Val Val

1235 1240 1245

Thr Tyr Val Asn Leu Thr Ser Asp Gln Leu Pro Asp Val Ile Pro

1250 1255 1260

Asp Tyr Ile Asp Val Asn Lys Thr Leu Asp Glu Ile Leu Ala Ser

1265 1270 1275

Leu Pro Asn Arg Thr Gly Pro Ser Leu Pro Leu Asp Val Phe Asn

1280 1285 1290

Ala Thr Tyr Leu Asn Leu Thr Gly Glu Ile Ala Asp Leu Glu Gln

1295 1300 1305

Arg Ser Glu Ser Leu Gln Asn Thr Thr Glu Glu Leu Arg Thr Leu

1310 1315 1320

Ile Tyr Asn Ile Asn Asn Thr Leu Val Asp Leu Glu Trp Leu Asn

1325 1330 1335

Arg Val Glu Thr Tyr Ile Lys Trp Pro Trp Trp Ile Trp Leu Ile

1340 1345 1350

Ile Phe Ile Val Leu Ile Phe Val Val Ser Leu Leu Val Phe Cys

1355 1360 1365

Cys Ile Ser Thr Gly Cys Cys Gly Cys Cys Gly Cys Cys Gly Ala

1370 1375 1380

Cys Phe Ser Gly Cys Cys Arg Gly Pro Arg Leu Gln Pro Tyr Glu

1385 1390 1395

Ala Phe Glu Lys Val His Val Gln Asp Ile Glu Pro Pro Thr Pro

1400 1405 1410

Ile Cys Pro Glu Ile Cys Ser Cys Pro Ala Ala Glu Val Leu Gly

1415 1420 1425

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Ile Leu

1430 1435 1440

Met Ile Ser Arg Thr Pro Lys Val Thr Cys Val Val Val Asp Val

1445 1450 1455

Ser Gln Glu Glu Ala Glu Val Gln Phe Ser Trp Tyr Val Asp Gly

1460 1465 1470

Val Gln Leu Tyr Thr Ala Gln Thr Arg Pro Met Glu Glu Gln Phe

1475 1480 1485

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gln His Gln

1490 1495 1500

Asp Trp Leu Lys Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys

1505 1510 1515

Asp Leu Leu Ser Pro Ile Thr Arg Thr Ile Ser Lys Ala Thr Gly

1520 1525 1530

Pro Ser Arg Val Pro Gln Val Tyr Thr Leu Pro Pro Ala Trp Glu

1535 1540 1545

Glu Leu Ser Lys Ser Lys Val Ser Ile Thr Cys Leu Val Thr Gly

1550 1555 1560

Phe Tyr Pro Pro Asp Ile Asp Val Glu Trp Gln Ser Asn Gly Gln

1565 1570 1575

Gln Glu Pro Glu Gly Asn Tyr Arg Thr Thr Pro Pro Gln Gln Asp

1580 1585 1590

Val Asp Gly Thr Tyr Phe Leu Tyr Ser Lys Leu Ala Val Asp Lys

1595 1600 1605

Val Arg Trp Gln Arg Gly Asp Leu Phe Gln Cys Ala Val Met His

1610 1615 1620

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser Lys Thr

1625 1630 1635

Gln Gly Lys

1640

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<213> artificial sequence

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Thr Gln Glu Val Tyr Asp Thr His Asp Cys Ala Thr Asn Gly Thr Ile

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Arg Pro Phe Lys Val Leu Ser Met Leu Glu Lys Cys Leu Thr Ala Gly

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Cys Tyr Ser Gln Leu Leu Ser Leu Trp Cys Ile Val Pro Phe Cys Phe

35 40 45

Ala Val Leu Val Asn Ala Ala Pro Lys Thr Ala Pro Ser Val Tyr Pro

50 55 60

Leu Ala Pro Cys Gly Arg Asp Val Ser Gly Pro Asn Val Ala Leu Gly

65 70 75 80

Cys Leu Ala Ser Ser Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn

85 90 95

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ser Val Leu Gln

100 105 110

Pro Ser Gly Leu Tyr Ser Leu Ser Ser Met Val Thr Val Pro Ala Ser

115 120 125

Ser Leu Ser Ser Lys Ser Tyr Thr Cys Asn Val Asn His Pro Ala Thr

130 135 140

Thr Thr Lys Val Asp Lys Arg Val Gly Ile His Gln Pro Gln Thr Cys

145 150 155 160

Pro Cys Pro Gly Cys Glu Val Ala Gly Pro Ser Val Phe Ile Phe Pro

165 170 175

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Gln Thr Pro Glu Val Thr

180 185 190

Cys Val Val Val Asp Val Ser Lys Glu His Ala Glu Val Gln Phe Ser

195 200 205

Trp Tyr Val Asp Gly Val Glu Val His Thr Ala Glu Thr Arg Pro Lys

210 215 220

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Ile

225 230 235 240

Gln His Gln Asp Trp Leu Lys Gly Lys Glu Phe Lys Cys Lys Val Asn

245 250 255

Asn Val Asp Leu Pro Ala Pro Ile Thr Arg Thr Ile Ser Lys Ala Ile

260 265 270

Gly Gln Ser Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Pro Ala Glu

275 280 285

Glu Leu Ser Arg Ser Lys Val Thr Leu Thr Cys Leu Val Ile Gly Phe

290 295 300

Tyr Pro Pro Asp Ile His Val Glu Trp Lys Ser Asn Gly Gln Pro Glu

305 310 315 320

Pro Glu Asn Thr Tyr Arg Thr Thr Pro Pro Gln Gln Asp Val Asp Gly

325 330 335

Thr Phe Phe Leu Tyr Ser Lys Leu Ala Val Asp Lys Ala Arg Trp Asp

340 345 350

His Gly Asp Lys Phe Glu Cys Ala Val Met His Glu Ala Leu His Asn

355 360 365

His Tyr Thr Gln Lys Ser Ile Ser Lys Thr Gln Gly Lys Tyr Val Leu

370 375 380

Ser Ser Ile Tyr Ala Val Cys Ala Leu Ala Ala Leu Thr Cys Phe Val

385 390 395 400

Ile Arg Phe Ala Lys Asn Cys Met Ser Trp Arg Tyr Ala Cys Thr Arg

405 410 415

Tyr Thr Asn Phe Leu Leu Asp Thr Lys Gly Arg Leu Tyr Arg Trp Arg

420 425 430

Ser Pro Val Ile Ile Glu Lys Arg Gly Lys Val Glu Val Glu Gly His

435 440 445

Leu Ile Asp Leu Lys Arg Val Val Leu Asp Gly Ser Val Ala Thr Pro

450 455 460

Ile Thr Arg Val Ser Ala Glu Gln Trp Gly Arg Pro

465 470 475

Claims (11)

1. A linker peptide consisting of SEQ ID NO:1, and a polypeptide comprising the amino acid sequence of 1.

2. A fusion protein, comprising: consists of SEQ ID NO:1, and a linker peptide consisting of the amino acid sequence of 1; and

an immunopotentiating substance attached to the C-terminus of the linker peptide.

3. The fusion protein according to claim 2, wherein the immunopotentiator is at least any one selected from the group consisting of an Fc region of an antibody, a flagellin, and interleukin-2.

4. The fusion protein of claim 2, wherein the immune enhancing substance is an Fc region of an antibody.

5. A polynucleotide encoding the fusion protein of claim 2.

6. A recombinant vector comprising the polynucleotide of claim 5.

7. A host cell transformed with the recombinant vector of claim 6.

8. A vaccine composition comprising: infectious virus-derived antigen; and

a fusion protein comprising a polypeptide consisting of SEQ ID NO:1 and an immunopotentiator attached to the C-terminus of said linker peptide.

9. The vaccine composition of claim 9, wherein the N-terminus of the connecting peptide is linked to an infectious virus-derived antigen.

10. The vaccine composition of claim 9, wherein the infectious virus-derived antigen is an antigen derived from porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, dengue virus, japanese encephalitis virus, zika virus, ebola virus, rotavirus, dengue virus, west nile virus, yellow fever virus, adenovirus, BK virus, smallpox virus, severe fever with thrombocytopenia syndrome virus, herpes simplex virus, EB virus, hepatitis a virus, hepatitis b virus, hepatitis c virus, hepatitis d virus, hepatitis e virus, hantavirus, or cytomegalovirus.

11. The vaccine composition of claim 9, wherein the vaccine composition is an attenuated live vaccine, an inactivated vaccine, a subunit vaccine, or a virus-like particle vaccine.