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WO2024253482A1 - Protéine recombinante comprenant une protéine de spicule de pedv s1 et une protéine dérivée de ferritine et utilisation associée - Google Patents

Protéine recombinante comprenant une protéine de spicule de pedv s1 et une protéine dérivée de ferritine et utilisation associée Download PDF

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WO2024253482A1
WO2024253482A1 PCT/KR2024/007861 KR2024007861W WO2024253482A1 WO 2024253482 A1 WO2024253482 A1 WO 2024253482A1 KR 2024007861 W KR2024007861 W KR 2024007861W WO 2024253482 A1 WO2024253482 A1 WO 2024253482A1
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protein
recombinant
pedv
ferritin
spike
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Korean (ko)
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신현진
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Industry and Academy Cooperation In Chungnam National University
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Industry and Academy Cooperation In Chungnam National University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A61K39/225—Porcine transmissible gastroenteritis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Porcine epidemic diarrhea is an infectious disease of pigs of all ages caused by porcine epidemic diarrhea virus (PEDV) infection, and is characterized by vomiting and watery diarrhea. This disease was first reported in Korea in 1992 and has since spread nationwide. It is the most damaging of all diarrheal diseases in suckling pigs and is a major disease that causes great economic damage to pig farms.
  • PEDV porcine epidemic diarrhea
  • a vaccine is an antigen used to actively immunize animals for the purpose of preventing infectious diseases, or a biological preparation containing an antigen as an effective ingredient, and refers to an immunogen that is administered to animals for the purpose of preventing infectious diseases and creates immunity in the body.
  • the body creates the corresponding antibodies and acquires immunity, and once created, the antibodies remain in the body for a relatively long time, so that even if infection by the pathogen of the corresponding disease occurs, it is possible to defend against it, and as a result, the disease can be prevented.
  • vaccines there are various types of vaccines, including killed vaccines that use bacteria, inactivated vaccines, live vaccines that use live bacteria, attenuated vaccines that use attenuated strains of live bacteria, bacterial toxoids or derivatives thereof, etc.
  • killed vaccines that use bacteria
  • inactivated vaccines live vaccines that use live bacteria
  • attenuated vaccines that use attenuated strains of live bacteria
  • bacterial toxoids or derivatives thereof etc.
  • One aspect provides a protein construct comprising the S1 protein of the PEDV (Porcine epidemic diarrhea virus) spike and a ferritin-derived protein.
  • PEDV Porcine epidemic diarrhea virus
  • Another aspect provides a vaccine composition for preventing or treating PEDV infection disease, comprising the protein structure as an active ingredient.
  • Another aspect provides a method for preventing or treating PEDV infection disease, comprising administering the vaccine composition to a subject other than a human.
  • One aspect is to provide a protein construct comprising a first recombinant protein in which the S1 protein of the PEDV (Porcine epidemic diarrhea virus) spike and a ferritin heavy chain protein are linked; and a second recombinant protein in which an antibody Fc region protein and a ferritin light chain protein are linked.
  • PEDV Porcine epidemic diarrhea virus
  • PEDV Porcine epidemic diarrhea virus
  • the genome of PEDV consists of a total of seven open reading frames (ORFs) encoding four structural proteins.
  • the structural proteins include a spike protein (S protein), a membrane protein, an envelope protein, and a nucleocapsid protein.
  • spike protein in this specification refers to a protruding structure arranged on the surface of a virus, with a length of 10 to 15 nm, which binds to protein receptors on the cell membrane and is responsible for penetrating our body. Therefore, the spike protein plays an important role when a virus enters a human cell. In particular, the host that a virus infects changes depending on the binding ability of the spike protein. Therefore, the spike protein can be utilized for the development of vaccines, antibodies, and diagnostics for virus treatment.
  • the spike protein of the PEDV is divided into a spike 1 (spike 1, S1) domain and a spike 2 (spike 2, S2) domain, among which S1 is a site involved in cell adhesion and contains a receptor binding site, and it is known that various neutralizing antibody-generating epitopes exist.
  • ferritin in this specification refers to a protein that stores iron and is widely found in prokaryotes and eukaryotes.
  • Ferritin has a molecular weight of approximately 500,000 Da, is composed of a heavy chain and a light chain, and has a unique property of self-assembly, forming spherical particles (e.g., a protein cage with an empty interior).
  • Ferritin is composed of 24 monomers (a single monomer or a heteromonomer composed of either a heavy chain or a light chain) to form a giant spherical tertiary structure.
  • the outer diameter of ferritin is approximately 12-13 nm and the inner diameter is approximately 7-8 nm.
  • Ferritin is harmless to the body, stable even at high temperatures (80-100°C) and in a wide range of hydrogen ion concentrations from pH 3 to 10, and has the advantages of ease of mass production and low production costs, so it can be applied as a vaccine carrier.
  • the ferritin-derived protein may be derived from porcine ferritin protein, and specifically may be derived from the light chain or the heavy chain of the ferritin protein.
  • antibody Fc region fragment crystallizable region
  • the term "antibody Fc region (fragment crystallizable region)" in this specification is located at the terminal part of the antibody and can activate the overall immune system through various mechanisms.
  • the antibody Fc region induces overall immune activation through antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC) through interaction with various immune cells and complement.
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cell-mediated phagocytosis
  • CDC complement-dependent cytotoxicity
  • various technologies such as fusion between antigen proteins and Fc regions are being applied, but are still inadequate.
  • protein structure refers to a spherical structure formed by self-assembly of the first recombinant protein and the second recombinant protein of the present invention.
  • the first recombinant protein may be one in which the S1 protein of the PEDV (Porcine epidemic diarrhea virus) spike and a ferritin heavy chain protein are linked/included
  • the second recombinant protein may be one in which an antibody Fc region protein and a ferritin light chain protein are linked/included.
  • the first recombinant protein and the second recombinant protein are each included in multiple units, for example, each of the recombinant proteins may act as a monomer.
  • the protein structure may be composed of a combination of about 24 to 30 monomers, for example, a combination of 24 to 30, 24 to 28, 24 to 26, 26 to 30, 26 to 28, or 28 to 30 monomers.
  • the S1 protein of the PEDV spike and the antibody Fc region protein in the protein structure may be positioned on the outer surface of the spherical structure.
  • the protein structure is provided in a form in which the S1 protein of the PEDV spike as an antigen and the antibody Fc region protein as an immune-enhancing component are fused as one body according to genetic design, thereby significantly improving immunogenicity.
  • the S1 protein of the PEDV spike and the ferritin heavy chain protein may be linked by a peptide linker, and the antibody Fc region protein and the ferritin light chain protein may also be linked by a peptide linker.
  • each of the recombinant proteins may further comprise a linker, and specifically, the linker may be positioned between the PEDV spike protein S1 derived protein/antibody Fc region protein and the ferritin derived protein (heavy chain/light chain).
  • the linker may be a peptide inserted between proteins so as to increase the structural flexibility of these proteins or to enhance the activity of each protein.
  • the linker is not limited as long as it does not inhibit the activity of each protein to be fused and does not cause an unnecessary immune response, but is preferably a peptide linker consisting of 1 to 20 amino acids, more preferably 1 to 5 amino acids, and specifically, may be a SSG peptide linker.
  • Conventional techniques utilizing the self-assembly properties based on ferritin have adopted a method of forming a self-assembly using ferritin and then linking effective ingredients/active components on the surface of the self-assembly, but this method has limitations in terms of quantification and stability of the effective ingredient.
  • by applying a genetic fusion method it was attempted to secure structural stability and ease of purification along with induction of an enhanced immune response.
  • the S1 protein and antibody Fc region protein of the above PEDV spike may be linked to the C-terminus or N-terminus of the ferritin heavy chain protein and the ferritin light chain protein, respectively, and specifically, may be linked to the C-terminus of a ferritin-derived protein.
  • the protein construct may include a first recombinant protein comprising a PEDV spike S1 protein having an amino acid sequence of SEQ ID NO: 1 and a ferritin heavy chain protein having an amino acid sequence of SEQ ID NO: 3, linked to each other; and a second recombinant protein comprising an antibody Fc region protein having an amino acid sequence of SEQ ID NO: 2 and a ferritin light chain protein having an amino acid sequence of SEQ ID NO: 4, linked to each other.
  • the protein construct may comprise a first recombinant protein comprising an amino acid sequence of SEQ ID NO: 5; and a second recombinant protein comprising an amino acid sequence of SEQ ID NO: 6.
  • the above protein structure or recombinant protein includes, without limitation, an amino acid sequence having an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 6, as well as an amino acid sequence having an amino acid sequence having an identity of 80% or more, specifically 90% or more, more specifically 95% or more, even more specifically 98% or more, and most specifically 99% or more with the above sequence, and which exhibits substantially the same or corresponding efficacy as the protein.
  • an amino acid sequence having such homology in which a part of the sequence is deleted, modified, substituted, or added, is also included within the scope of the present invention.
  • homology in this specification refers to the degree of similarity between a base sequence encoding a protein or an amino acid sequence constituting a protein. If the homology is sufficiently high, the expression product and protein of the corresponding gene may have the same or similar activity. In addition, the homology may be expressed as a percentage according to the degree of matching with a given amino acid sequence or base sequence.
  • a homologous sequence having the same or similar activity as a given amino acid sequence or nucleotide sequence is expressed as "% homology.”
  • the sequences can be compared using standard software that calculates parameters such as score, identity and similarity, specifically BLAST 2.0, or by hybridization experiments performed under defined stringent conditions, and the defined appropriate hybridization conditions are within the scope of the art and can 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).
  • Another aspect is to provide a polynucleotide encoding the recombinant protein of the present invention.
  • the same parts as described above also apply to the polynucleotide.
  • polynucleotide in this specification refers to DNA, a polymeric substance in which nucleotides are bound and which encodes genetic information.
  • the polynucleotide may include a coding sequence for a first recombinant protein comprising a PEDV spike S1 comprising a nucleotide sequence of SEQ ID NO: 7 and a ferritin heavy chain region comprising a nucleotide sequence of SEQ ID NO: 9; and a coding sequence for a second recombinant protein comprising an antibody Fc region comprising a nucleotide sequence of SEQ ID NO: 9 and a ferritin light chain region comprising a nucleotide sequence of SEQ ID NO: 10.
  • the polynucleotide may comprise a coding sequence for a first recombinant protein comprising a nucleotide sequence of SEQ ID NO: 11; and a coding sequence for a second recombinant protein comprising a nucleotide sequence of SEQ ID NO: 12.
  • the polynucleotide encoding the above proteins can have various modifications in the coding region within a range that does not change the amino acid sequence of the protein expressed from the coding region, considering the codon preferred in the organism that is to express the above protein due to the degeneracy of the codon. Therefore, the polynucleotide can include without limitation any base sequence encoding each protein.
  • a probe that can be prepared from a known sequence for example, a sequence that hybridizes under strict conditions with a complementary sequence to all or part of the above polynucleotide sequence and encodes a protein having the same activity as the above protein, can be included without limitation.
  • Hybridization requires that the two polynucleotides have complementary sequences, although mismatches between bases are possible depending on the stringency of hybridization.
  • complementary is used to describe the relationship between nucleotide bases that are capable of hybridizing with each other.
  • adenosine is complementary to thymine
  • cytosine is complementary to guanine.
  • the present application may also encompass isolated polynucleotide fragments that are complementary to the entire sequence, as well as substantially similar polynucleotide sequences.
  • polynucleotides having homology can be detected using hybridization conditions including a hybridization step at a Tm value of 55° C. and using the conditions described above.
  • the Tm value may be, but is not limited to, 60° C., 63° C. or 65° C. and may be appropriately adjusted by a person skilled 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 the variables are well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).
  • expression vector refers to a recombinant vector capable of expressing a target protein when introduced into a suitable host cell, and a genetic construct including essential regulatory elements that are operably linked to allow the gene insert to be expressed.
  • operably linked means that a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein are functionally linked to perform a general function.
  • the operably linked vector can be produced using a genetic recombination technique well known in the art, and site-specific DNA cleavage and ligation can be easily performed using enzymes, etc. that are generally known in the art.
  • Suitable expression vectors of the present invention may include, in addition to expression control elements such as a promoter, an initiation codon, a stop codon, a polyadenylation signal and an enhancer, a signal sequence for membrane targeting or secretion.
  • the initiation codon and the stop codon are generally considered to be part of the nucleotide sequence encoding the immunogenic target protein, and must be functional in a subject when the genetic construct is administered and must be in frame with the coding sequence.
  • Common promoters can be constitutive or inducible and include, but are not limited to, the lac, tac, T3 and T7 promoters in prokaryotes, and the simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoters, human immunodeficiency virus (HIV), e.g. the long terminal repeat (LTR) promoter of HIV, Moloney virus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), Rous sarcoma virus (RSV) promoters, as well as the ⁇ -actin promoter, human hemoglobin, human muscle creatine and promoters derived from human metallothionein, in eukaryotes.
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HAV human immunodeficiency virus
  • LTR long terminal repeat
  • CMV cytomegalovirus
  • EBV Epstein-Barr virus
  • RSV Rous sarcoma virus
  • the expression vector may include a selectable marker for selecting a host cell containing the vector.
  • the selectable marker is for selecting cells transformed with the vector, and markers that confer a selectable phenotype such as drug resistance, nutrient requirement, resistance to cytotoxic agents, or expression of surface proteins may be used. In an environment treated with a selective agent, only cells expressing the selectable marker survive, so transformed cells can be selected.
  • the vector may include a replication origin, which is a specific nucleic acid sequence where replication is initiated.
  • recombinant expression vectors for inserting foreign genes.
  • the type of recombinant vector is not particularly limited as long as it has the function of expressing a desired gene and producing a desired protein in various host cells of prokaryotic and eukaryotic cells.
  • a vector that has a promoter that exhibits strong activity and a strong expression ability while being able to mass-produce a foreign protein in a form similar to that in the natural state can be used.
  • Expression vectors that can be used in bacterial hosts include, but are not limited to, bacterial plasmids obtained from Escherichia coli, such as pcDNA3.1, pET, pRSET, pBluescript, pGEX2T, pUC vector, col E1, pCR1, pBR322, pMB9 or derivatives thereof, plasmids having a wider host range such as RP4, phage DNA such as phage lambda derivatives such as ⁇ gt10, ⁇ gt11 or NM989, and other DNA phages such as M13 and filamentous single-stranded DNA phages.
  • Yeast cells can be used, for example, 2°C plasmid or derivatives thereof, and insect cells can be used, for example, pVL941.
  • Another aspect is to provide a vaccine composition for preventing or treating PEDV infection disease, comprising the protein structure of the present invention as an active ingredient.
  • a vaccine composition for preventing or treating PEDV infection disease comprising the protein structure of the present invention as an active ingredient.
  • the same parts as described above are also applied to the composition.
  • the term "vaccine” as used herein means a pharmaceutical composition containing at least one immunologically active component that induces an immunological response in an animal.
  • the immunologically active component of the vaccine may contain suitable elements of live or killed viruses (subunit vaccines), whereby these elements are prepared by a process of synthesis induced by appropriate manipulation of a suitable system, such as but not limited to bacteria, insects, mammals or other species, followed by isolation and purification, or by direct introduction of genetic material into an animal in need of the vaccine using a suitable pharmaceutical composition (polynucleotide vaccination).
  • the vaccine may contain one or more of the above-described elements at the same time.
  • prevention in this specification means any act of inhibiting or delaying infection with PEDV and the onset of disease caused by said infection by administration of a PEDV vaccine composition.
  • treatment in this specification means any action that improves or benefits the symptoms of a disease already caused by infection with PEDV due to administration of a PEDV vaccine composition.
  • the above vaccine composition may further comprise a pharmaceutically acceptable excipient, diluent or carrier.
  • pharmaceutically acceptable excipient, diluent or carrier may mean an excipient, diluent or carrier that does not stimulate a living organism and does not inhibit the biological activity and properties of the compound to be injected.
  • pharmaceutically acceptable means that it does not inhibit the activity of the active ingredient and does not have toxicity that is more than the target of application (prescription) can adapt to.
  • Suitable carriers for vaccines are known to those skilled in the art and include, but are not limited to, proteins, sugars, etc.
  • the carriers may be aqueous solutions, non-aqueous solutions, suspensions, or emulsions.
  • Structured or amorphous organic or inorganic polymers may be used as immunoadjuvants to increase immunogenicity.
  • Immunoadjuvants are generally known to promote immune responses through chemical and physical binding to antigens.
  • Amorphous aluminum gels, oil emulsions, double oil emulsions, and immunosols may be used as immunoadjuvants.
  • various plant-derived saponins, levamisole, CpG dinucleotides, RNA, DNA, LPS, various types of cytokines, etc. may be used to promote immune responses.
  • Such immune compositions may be used as compositions for inducing optimal immune responses by combining various adjuvants and immune response-promoting additives.
  • stabilizers, inactivators, antibiotics, preservatives, etc. may be used as compositions that can be added to the vaccine.
  • the vaccine antigen can also be mixed with distilled water, buffer solution, etc.
  • the above vaccine composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or unit dose ampoules or multiple dose injections according to conventional methods.
  • oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or unit dose ampoules or multiple dose injections according to conventional methods.
  • diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, or surfactants that are generally used.
  • Another aspect provides a method for preventing or treating a PEDV infection disease, comprising administering the vaccine composition to a non-human subject.
  • the same parts as described above also apply to the method.
  • subject means a living organism that can be infected with PEDV and can develop a disease due to the infected PEDV, preferably a mammal, but is not particularly limited thereto.
  • the mammal may include a cow, a horse, a sheep, a pig, a goat, a camel, an antelope, a dog, a cat, a rat, livestock, etc., and may be specifically a pig.
  • administration means introducing a given substance into an individual by an appropriate method, and the route of administration of the vaccine composition of the present invention may be administered through any common route as long as it can reach the target tissue. It may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, topically, intranasally, intrapulmonary, or rectally, but is not limited thereto. However, when administered orally, since proteins are digested, it is preferable that the oral composition be formulated to coat the active agent or protect it from decomposition in the stomach. In addition, the pharmaceutical composition may be administered by any device through which the active agent can travel to the target cell.
  • the above vaccine composition can be administered in a pharmaceutically effective amount, wherein the term "pharmaceutically effective amount” means an amount sufficient to treat or prevent a disease at a reasonable benefit/risk ratio applicable to medical treatment or prevention, and the effective dosage level can be determined according to the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and the excretion rate, the treatment period, the drug used in combination or simultaneously with the composition of the present invention used, and other factors well known in the medical field.
  • the vaccine composition can be administered alone or in combination with a component known to exhibit a preventive or therapeutic effect against a known PEDV infectious disease. It is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects by considering all of the above factors.
  • the dosage of the above vaccine composition can be determined by a person skilled in the art in consideration of the purpose of use, the degree of toxicity of the disease, the patient's age, weight, sex, medical history, or the type of substance used as the effective ingredient.
  • the vaccine composition of the present invention can be administered to an adult at about 0.1 ng to about 1,000 mg/kg, preferably 1 ng to about 100 mg/kg, and the frequency of administration of the composition of the present invention is not particularly limited thereto, but can be administered once a day or administered several times in divided doses.
  • the dosage or frequency of administration does not limit the scope of the present invention in any way.
  • the above vaccine composition can be administered as an individual treatment or in combination with other treatments, and can be administered sequentially or simultaneously with conventional treatments. And it can be administered singly or multiple times. It is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects by considering all of the above factors, and this can be easily determined by those skilled in the art.
  • a recombinant protein can provide a ferritin-based self-assembling structure containing the S1 protein of the PEDV spike, which is an antigen protein, and a porcine-derived antibody Fc region protein.
  • the structure can induce an enhanced immune response to the PEDV spike protein, and thus can be utilized as an effective ingredient in a vaccine composition.
  • Figure 1 is a schematic diagram showing the structure of a recombinant protein and protein structure according to one embodiment.
  • Figure 2 shows the results of Western blot analysis to confirm the expression of the first recombinant protein (PS1-FTHC) according to one embodiment.
  • Figure 3 shows the results of Western blot analysis to confirm the expression of the second recombinant protein (sFc-FTLC) according to one embodiment.
  • Figure 4 shows the results of Western blot analysis to confirm the simultaneous expression of the first recombinant protein and the second recombinant protein (PS1/sFC-FT) according to one embodiment.
  • Figure 5 shows the results of confirming the expression of the first recombinant protein (PS1-FTHC) according to one embodiment through Western blot and confirming the protein structure formed by self-assembly through electron microscopy.
  • Figure 7 shows the results of confirming the expression level of a recombinant protein during the process of purifying and concentrating a protein structure according to one embodiment.
  • FIG. 8 shows the results of confirming the expression level of a recombinant protein under reducing or non-reducing conditions after performing the process of purifying and concentrating a protein structure according to one embodiment.
  • FIG. 9 is a result of observing the generated spherical protein structures using an electron microscope and quantifying and evaluating their size distribution after performing a process of purifying and concentrating a protein structure according to one embodiment.
  • Figure 10 shows the results of quantitatively evaluating the level of formation of specific antibodies against the PEDV spike protein 14 days after inoculation of a protein construct according to one embodiment into mice.
  • Figure 11 shows the results of quantitatively evaluating the level of formation of specific antibodies against the PEDV spike protein 40 days after inoculation of a protein construct according to one embodiment into mice.
  • Figure 12 shows the results of quantitatively evaluating the level of formation of neutralizing antibodies against the PEDV spike protein 14 days after inoculation of a protein construct according to one embodiment into mice.
  • Figure 13 shows the results of quantitatively evaluating the level of formation of neutralizing antibodies against the PEDV spike protein 40 days after inoculation of a protein construct according to one embodiment into mice.
  • the S1 protein (subunit) in the spike of PEDV was used as an antigen peptide capable of inducing a vaccine response against PEDV, and a porcine antibody Fc region protein was used as a component for inducing an enhanced immune response.
  • a first recombinant protein in which the PEDV S1 protein and the ferritin heavy chain protein were linked, and a second recombinant protein in which the Fc region protein and the ferritin light chain protein were linked were designed, and an SSG protein linker was used for linking the proteins.
  • a 6 His tag was positioned in the N-terminal region, and a flag sequence was positioned in the C-terminal region of the S1 spike protein of PEDV, and in the second recombinant protein, a Myc tag was positioned in the C-terminal region of the ferritin light chain protein.
  • the specific structures of the first recombinant protein and the second recombinant protein are as shown in Fig. 1.
  • information on the amino acid sequences of the recombinant proteins manufactured in this example is as shown in Table 1, and information on the nucleotide sequences encoding the proteins are shown in SEQ ID NOs: 7 to 12, respectively.
  • Table 1 the amino acid sequence encoded by the initiation codon is underlined.
  • a polynucleotide encoding the protein was cloned and introduced into a pCDNA 3.1 myc his vector or a pCAGGS vector to produce a recombinant vector. Thereafter, the vector was transfected into 293T cells or IPEC (Porcine intestinal epithelial cell) cells, respectively, according to a conventional method known in the art, and the transfected cells were cultured, thereby producing the first and second recombinant proteins, respectively.
  • the produced recombinant proteins structurally all contain ferritin protein as a monomer.
  • the pCDNA 3.1 myc his vector into which the polynucleotide described above was introduced was transfected into 293T cells. Thereafter, the lysate of the transfected cells was subjected to Western blotting using antigen beads, flag antibodies, and Myc antibodies to confirm whether the S1 protein of the PEDV spike and the antibody Fc region were expressed.
  • the first recombinant protein (PS1-FTHC) and the second recombinant protein (sFC-FTLC) were simultaneously expressed, respectively.
  • the first recombinant protein (PS1-FTHC) was effectively expressed, and the formation of a spherical structure by self-assembly of the monomers was confirmed.
  • FIG. 6 it was confirmed that, similarly to the above, the first recombinant protein (PS1-FTHC) and the second recombinant protein (sFC-FTLC) were simultaneously expressed.
  • each plasmid construct (PEDV S1 and PEDV S1/sFC FT) was transfected into 293T cells (15 ⁇ g/10 cm dish) and cultured for 3 days for recombinant protein expression.
  • Cells were harvested using PBS and the cell pellet was suspended in M-per lysis buffer (Invitrogen). 1 mL of lysis buffer was used per dish. The lysed cells were centrifuged to recover the protein extract. Then, 5 mL of the protein extract was loaded onto a His affinity resin-containing column (ROCHE) in the presence of 10 mM imidazole at pH 7.6 and passed through at a speed of 1 mL/1 min.
  • ROCHE His affinity resin-containing column
  • the column was washed with 15 mL of wash buffer (20 mM Tris-HCl and 50 mM NaCl at pH 7.5, 20 mM imidazole).
  • the recombinant ferritin complex was eluted using 10 mL of elution buffer (20 mM Tris-HCl and 50 mM NaCl, 200 mM Imidazole).
  • the eluted protein was concentrated using a 100 kDa Amicon centrifugal filter unit. The protein concentration was measured using BCA.
  • the extracted spherical protein structures were then observed by electron microscopy, and their size distribution was quantified.
  • a protein construct according to one embodiment to form specific antibodies and neutralizing antibodies against the PEDV spike protein was confirmed.
  • PBS group administered only PBS
  • PEDB-S1/sFC FT group administered a protein construct according to one embodiment
  • each group of mice was inoculated into the anterior proximal quadriceps muscle (intramuscularly) with 100 ⁇ L of protein or PBS.
  • additional inoculations were performed twice every two weeks (days 14 and 28).
  • the third vaccination day 42, 60
  • blood was collected from the orbit of the mice.
  • the level of specific antibody formation was measured by ELISA (Enzyme-Linked Immunosorbent Assay) on the collected blood (serum), and the relative level was compared based on the degree of PEDV S1 production.
  • the level of neutralizing antibody formation was additionally compared for the same control and experimental groups as above.

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Abstract

La présente invention concerne : une protéine recombinante comprenant une protéine S1 de spicule du virus de la diarrhée épidémique porcine (PEDV) et une protéine dérivée de la ferritine ; et une utilisation de celles-ci, et une structure protéique produite à partir de la protéine recombinante, une composition vaccinale comprenant la structure protéique, et une méthode de prévention ou de traitement de maladies infectieuses PEDV, la méthode comprenant une étape d'administration de la composition vaccinale à un sujet non humain.
PCT/KR2024/007861 2023-06-09 2024-06-10 Protéine recombinante comprenant une protéine de spicule de pedv s1 et une protéine dérivée de ferritine et utilisation associée Pending WO2024253482A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20160132494A (ko) * 2014-04-03 2016-11-18 베링거잉겔하임베트메디카인코퍼레이티드 돼지 유행성 설사 바이러스 백신
US20170202951A1 (en) * 2014-07-11 2017-07-20 Zoetis Services Llc Novel Vaccine Compositions for Porcine Epidemic Diarrhea Virus and Porcine Deltacoronavirus
WO2018081318A1 (fr) * 2016-10-25 2018-05-03 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Protéines de spicule de coronavirus de préfusion et utilisation associée
KR20220133632A (ko) * 2021-03-25 2022-10-05 충남대학교산학협력단 Pedv 스파이크 단백질 s1 유래 단백질 및 페리틴 유래 단백질을 포함하는 재조합 단백질 및 이의 용도
KR20220136057A (ko) * 2021-03-31 2022-10-07 충남대학교산학협력단 바이러스 표면 엔지니어링 기반의 면역 증강된 바이러스 백신

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20160132494A (ko) * 2014-04-03 2016-11-18 베링거잉겔하임베트메디카인코퍼레이티드 돼지 유행성 설사 바이러스 백신
US20170202951A1 (en) * 2014-07-11 2017-07-20 Zoetis Services Llc Novel Vaccine Compositions for Porcine Epidemic Diarrhea Virus and Porcine Deltacoronavirus
WO2018081318A1 (fr) * 2016-10-25 2018-05-03 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Protéines de spicule de coronavirus de préfusion et utilisation associée
KR20220133632A (ko) * 2021-03-25 2022-10-05 충남대학교산학협력단 Pedv 스파이크 단백질 s1 유래 단백질 및 페리틴 유래 단백질을 포함하는 재조합 단백질 및 이의 용도
KR20220136057A (ko) * 2021-03-31 2022-10-07 충남대학교산학협력단 바이러스 표면 엔지니어링 기반의 면역 증강된 바이러스 백신

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LEE, D. -K. ET AL.: "The N-terminal Region of the Porcine Epidemic Diarrhea Virus Spike Protein is Important for the Receptor Binding", KOREAN J. MICROBIOL. BIOTECHNOL., vol. 39, no. 2, 2011, pages 140 - 145, XP008177257 *

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