KR20230004330A - Peptide for enhancing neutralizing antibody-producing ability and vaccine composition comprising the same - Google Patents

Abstract

The present invention relates to a novel peptide capable of enhancing the ability to form neutralizing antibodies and uses thereof. The vaccine composition comprising an antigen and the peptide derived from the spike protein of middle east respiratory syndrome (MERS) coronavirus of the present invention is superior to other vaccines in forming neutralizing antibodies, and thus has the advantage of being able to manufacture a vaccine with improved efficacy.

Description

Peptide for enhancing neutralizing antibody-producing ability and vaccine composition comprising the same {Peptide for enhancing neutralizing antibody-producing ability and vaccine composition comprising the same}

The present invention relates to a novel peptide capable of enhancing the ability to form neutralizing antibodies and its use.

Vaccine is an antigen used to actively immunize animals for the prevention of infection, or a biological agent containing the antigen as an active ingredient, proposed by French microbiologist L. Pasteur. In particular, a vaccine as a biological preparation refers to an immunogen that is administered to an animal for the purpose of preventing an infectious disease and gives immunity to a living body. Normally, when a vaccine is administered, immunity is acquired by making the corresponding antibody in the living body, and once produced, the antibody remains in the living body for a relatively long time, so even if infection by the causative agent of the disease occurs, it is possible to defend against it and consequently prevent the disease. It can.

In general, vaccine preparations include killed vaccines that kill bacteria, inactivated vaccines, live vaccines that use live bacteria as they are, and attenuated strains that weaken live bacteria. There are various types of vaccines, such as attenuated vaccines, bacterial toxoids, or derivatives thereof. To develop an effective vaccine, it is necessary to facilitate the formation of antibodies against disease-causing agents so that the in vivo immune response is appropriate. Since it must be induced, it is desirable to develop it in a form similar to the possible disease causative agent.

On the other hand, adjuvants play an important role in the prevention and treatment of infectious diseases by activating immune cells. In general, adjuvant components refer to materials and methods that regulate (enhance or suppress) the immune response, and enhance or non-specifically increase or decrease the immune response by acting on the antigen administration form and immune-competent cells. Refers to an inhibitory substance. As a substance with regulatory activity centered on strengthening of the immune response, bacterial cells such as BCG and Propionibacterium acnes, cell walls, bacterial cell components such as trehalose dimycolate (TDM), and lipopolysaccharide (LPS), which is an endotoxin of Gram-negative bacteria In addition to synthetic compounds such as lipid A fraction, β-glucan (polysaccharide), N-acetylmuramyl dipeptide (MDP), bestatin, and levamisole, proteins derived from biological components such as thymus hormone, thymus factor, and taftin Peptidic substances are known.

In addition, lymphokine, monokine, interferon, and cyclophosphamide, which have regulatory activities such as enhancement of immune response by removing suppressive T cells and suppressive macrophages, are also treated as immune adjuvant components in a broad sense. . In addition, as an adjuvant for the antigen administration form, Freund's incomplete adjuvant (FIA), which is formulated as a water-in-oil type emulsion by mixing equal amounts of mineral oil including Arlacel A and an aqueous antigen solution, is used as an adjuvant component.

These immune adjuvant components not only enhance but also suppress the immune response depending on the route of administration, dosage, and timing of administration, and also, depending on the type of adjuvant, produce antibodies in the blood against antigens, induce cellular immunity, and suppress immunoglobulins. It is known that there are differences between classes.

Under this background, the present inventors developed a peptide derived from the spike protein of MERS (Middle East Respiratory Syndrome) coronavirus that can enhance the ability to form neutralizing antibodies and a vaccine composition containing the same, and verified the excellent ability of the vaccine to form neutralizing antibodies Thus, the present invention was completed.

One aspect is 1) an antigen; and 2) a peptide derived from a spike protein of MERS (Middle East Respiratory Syndrome) coronavirus or a polynucleotide encoding the same.

Another aspect provides a method for preventing or treating bacterial, viral and/or microbial infections and infectious diseases comprising administering the vaccine composition to an individual.

Another aspect provides an immune adjuvant or immune enhancer composition for a vaccine comprising a peptide derived from a spike protein of MERS (Middle East Respiratory Syndrome) coronavirus or a polynucleotide encoding the same.

Another aspect provides a peptide for enhancing neutralizing antibody formation ability, including a peptide derived from a spike protein of MERS (Middle East Respiratory Syndrome) coronavirus.

Another aspect provides a polynucleotide encoding the peptide for enhancing the ability to form neutralizing antibodies of the present invention.

One aspect is 1) an antigen; and 2) an immune enhancing peptide derived from a spike protein of MERS (Middle East Respiratory Syndrome) coronavirus or a polynucleotide encoding the same.

As used herein, the term "vaccine" is a biological preparation containing an antigen that gives immunity to a living body, and is an immunogen or antigenic substance that gives immunity to a living body by administering to a human or animal for the prevention of infection and / or infection. say

The vaccine may be in the form of a killed vaccine, an attenuated vaccine, a subunit vaccine, a conjugate vaccine, a recombinant vaccine, a monovalent vaccine, a multivalent vaccine or a combination vaccine.

The term "antigen" used herein refers to a substance that induces an immune response in a recipient, and in the present invention, a substance exhibiting an immune response inducing effect may be used without limitation.

The antigen may be selected from the group consisting of peptides, proteins, nucleic acids, sugars, pathogens, attenuated pathogens, inactivated pathogens, viruses, virus-like particles (VLPs), cells and cell fragments.

The antigens are coronavirus antigens, Japanese encephalitis virus antigens, HIB (Heamophilus influenzae type B) antigens, Zika virus antigens, Pseudomonas aeruginosa antigens, pertussis antigens, and tuberculosis antigens. , anthrax antigen, HAV (Hepatitis A virus) antigen, HBV (Hepatitis B virus) antigen, HCV (Hepatitis C virus) antigen, HIV (human immunodeficiency virus) antigen, HSV (Herpes simplex virus) antigen, age Antigens of Neisseria meningitidis, antigens of Corynebacterium diphtheria, antigens of Bordetella pertussis, antigens of Clostridium tetani, human papilloma (HPV) virus, Varicella virus, Enterococci antigen, Staphylococcus aureus antigen, Klebsiella pneumonia antigen, Acinetobacter baumannii antigen , Enterobacter antigen, Helicobacter pylori antigen, malaria antigen, dengue virus antigen, Orientia tsutsugamushi antigen, severe fever with thrombocytopenia syndrome Bunyavirus; SFTS Bunyavirus ), an influenza virus antigen, an Ebola virus antigen, and a pneumococcal antigen.

As used herein, the term "Coronavirus" refers to a virus belonging to the Family Coronaviridae and having an RNA genome surrounded by an outer membrane. The coronavirus is named after the crown-like shape of surface spike proteins, and has positive-strand RNA as its genome. This RNA contains information such as nucleocapsid (N), envelope (E), membrane (membrane: M), and surface spike (S) proteins that encode the structure of the virus. In coronaviruses, the receptor binding domain (RBD) of the surface projection (S) protein binds to and fuses with the host cell's receptor, penetrating into the host and performing self-replication.

The coronavirus may be selected from the group consisting of alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus, specifically porcine epidemic diarrhea virus (PEDV), canine coronavirus : CCV), feline infectious peritonitis virus (FIPV), bovine coronavirus (BCoV or BCV), avian infectious bronchitis virus (IBV), and transmissible gastroenteritis coronavirus : TGEV), severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (Middle East respiratory syndrome coronavirus) East respiratory syndrome coronavirus: MERS-CoV), or a combination thereof. SARS-CoV-2 may be a major cause of coronavirus disease 2019 (COVID-19).

The coronavirus infectious disease includes not only coronavirus infection, but also any symptoms caused by it, including fever, cough, dyspnea, sore throat, headache, muscle pain, and pneumonia, and complications caused by it. Specifically, the coronavirus infection diseases include coronavirus cold, coronavirus enteritis, coronavirus diarrhea, coronavirus pneumonia, severe acute respiratory syndrome (SARS), middle east respiratory syndrome : MERS), coronavirus disease 2019 (COVID-19), acute respiratory distress syndrome (ARDS), avian infectious bronchitis, or a combination thereof.

The vaccine may be for preventing or treating the bacterial and/or viral infection or infectious disease.

The term "prevention" in the present specification refers to all activities that suppress or delay infection of the bacteria and/or virus and onset of diseases caused by the infection due to administration of the vaccine composition.

The term "treatment" in the present specification refers to any action that improves or benefits the symptoms of a disease already caused by the bacterial and/or viral infection due to administration of the vaccine composition.

The vaccines are coronavirus vaccine, Japanese encephalitis vaccine, Hemophilus influenzae type B vaccine, MERS vaccine, Zika vaccine, Pseudomonas aeruginosa vaccine, cancer vaccine, tuberculosis vaccine, anthrax vaccine, HAV vaccine, HBV vaccine, HCV vaccine, HIV vaccine vaccine, herpes simplex vaccine, meningitis vaccine, diphtheria vaccine, whooping cough vaccine, tetanus vaccine, varicella vaccine, multidrug-resistant bacteria vaccine, enterococcal vaccine, staphylococcal vaccine, pneumococcal vaccine, acinetobacter vaccine, enterobacter vaccine, helicobacter vaccine, It may be selected from the group consisting of malaria vaccine, dengue fever vaccine, tsutsugamushi vaccine, severe fever with thrombocytopenia syndrome vaccine, SARS vaccine, Ebola vaccine, influenza vaccine, Corona 19 vaccine and pneumococcal vaccine.

The vaccine composition may enhance the ability to form neutralizing antibodies formed by an antigen, and specifically, the peptide derived from the spike protein of the MERS coronavirus may enhance the ability to form neutralizing antibodies.

As used herein, the term "neutralizing antibody" means an antibody that neutralizes the biological effects of pathogens or infectious particles when they penetrate the body to defend cells. Neutralizing antibodies are part of the immune response of the acquired immune system against viruses, intracellular bacteria and microbial toxins. Neutralizing antibodies are produced and bound to the surface structure of infectious particles in a specialized form to prevent the interaction of infectious antigens with host cells to achieve immunity, and this reaction is called antibody neutralization. In general, when a vaccine is administered, general antibodies and neutralizing antibodies are produced in the body, and general antibodies cause a general immune response, and neutralizing antibodies cause an immune response against a specific antigen. Therefore, it can be considered as an effective vaccine when the number of neutralizing antibodies is higher than that of normal antibodies.

Promoting or improving the neutralizing antibody formation ability/capability may be to improve the neutralizing antibody formation ability by improving the immune function in vivo by binding the peptide derived from the spike protein of the MERS coronavirus to DPP4/CD26 in vivo.

The peptide derived from the spike protein of the MERS coronavirus may be used as an immune enhancer or adjuvant.

The term "adjuvant" as used herein refers to a substrate or additive that cannot induce specific immunity to a vaccine antigen/immunogen by itself, but acts to increase an immune response by stimulating the immune system when working with an antigen. . That is, a vaccine using an antigen and an adjuvant together induces a stronger immune response than that induced by the antigen alone.

The peptide derived from the spike protein of the MERS coronavirus may include the amino acid sequence of SEQ ID NO: 1 as an immune enhancing peptide, and may specifically consist of the amino acid sequence of SEQ ID NO: 1.

The polynucleotide encoding the peptide derived from the spike protein of the MERS coronavirus may include the nucleotide sequence of SEQ ID NO: 2, and specifically may be composed of the nucleotide sequence of SEQ ID NO: 2.

The vaccine composition or immune enhancing peptide may further include a signal peptide or a polynucleotide encoding the same. The signal peptide may be a signal peptide derived from Gaussian luciferase or a variant thereof.

As used herein, the term "signal peptide" refers to a short peptide (5 to 30 amino acids) present at the N-terminus of a newly synthesized protein so that it is secreted to a designated location through a secretory pathway. ) means The signal peptide may be used interchangeably with 'signal peptide' or 'secretory peptide'.

The signal peptide derived from Gaussian luciferase may include the amino acid sequence of SEQ ID NO: 11, and may specifically consist of the amino acid sequence of SEQ ID NO: 11. In addition, an amino acid sequence exhibiting 80% or more, specifically 90% or more, more specifically 95% or more, more specifically 98% or more, and most specifically 99% or more homology with the sequence, substantially the signal Any amino acid sequence of a protein exhibiting the same or equivalent efficacy as that of the peptide is included without limitation.

In addition, the signal peptide derived from the Gaussian luciferase is a conservative substitution in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids in the amino acid sequence of SEQ ID NO: 11 It may, but is not limited thereto.

The term "conservative substitution" as used herein refers to the substitution of one amino acid with another amino acid having similar structural and/or chemical properties. The signal peptide derived from the Gaussian luciferase may have, for example, one or more conservative substitutions while still retaining the biological activity of the native or unmutated signal peptide. Such amino acid substitutions can generally occur based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; Negatively charged (acidic) amino acids include glutamic acid and aspartic acid; Aromatic amino acids include phenylalanine, tryptophan and tyrosine; Hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan. In addition, amino acids can be classified into amino acids with electrically charged side chains and amino acids with uncharged side chains. Amino acids with charged side chains include aspartic acid, glutamic acid, lysine, arginine, and histidine, and amino acids with non-charged side chains can be further classified as nonpolar amino acids or polar amino acids. Non-polar amino acids are glycine, alanine, valine, leucine, and isoleucine. contains methionine, proline; Polar amino acids may include serine, threonine, cysteine, asparagine, and glutamine. Conservative substitutions with amino acids having similar properties as described above can be expected to exhibit the same or similar activity.

The variant of the signal peptide derived from the Gaussian luciferase may be one in which 1 to 5 amino acids are added to the C-terminus of the signal peptide wild-type sequence, specifically 1 to 5, 1 to 4, 1 2 to 3, 2 to 5, 2 to 4, 2 to 3, 3 to 5, or 3 to 4 may be added. In addition, the amino acid added to the variant may be glycine (G), alanine (A), or a combination thereof.

The variant may be one in which 1 to 4 glycines (G) and 1 to 4 alanines (A) are added to the C-terminus of the wild-type signal peptide derived from Gaussian luciferase. Specifically, 1 glycine (G) and 1 to 4 alanines (A) may be added.

The variant may be one in which the amino acid sequences of G, G-A, G-A-A, G-A-A-A and G-A-A-A-A are added to the C-terminus of the signal peptide derived from Gaussian luciferase, specifically G-A-A (glycine-alanine-alanine) at the C-terminus. may have been added.

The variant may include one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 12 to 16, and specifically, one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 12 to 16. In addition, the amino acid sequences of SEQ ID NOs: 12 to 16 may be those in which an amino acid sequence of G, G-A, G-A-A, G-A-A-A or G-A-A-A-A is added to the C-terminus of the Gaussian luciferase-derived signal peptide composed of the amino acid sequence of SEQ ID NO: 11, respectively. . More specifically, the signal peptide may include the amino acid sequence of SEQ ID NO: 14.

The polynucleotide encoding the wild-type signal peptide derived from Gaussian luciferase may include or consist of the sequence of SEQ ID NO: 17. The polynucleotide sequence encoding the variant may include one or more selected from the group consisting of SEQ ID NOs: 18 to 22, or may be composed of one or more of the above sequences. In addition, when the encoding sequence is an RNA sequence, T in the polynucleotide sequences of SEQ ID NOs: 17 to 22 may be substituted with U.

The vaccine composition may include a fusion protein in which the antigen protein and the peptide are linked or a polynucleotide encoding the fusion protein. In addition, the vaccine composition may include a fusion protein or a polynucleotide encoding the fusion protein linked to at least one selected from the group consisting of the antigen protein, the immune enhancing peptide, and the signal peptide.

The fusion protein may include the antigen protein, the signal peptide, and/or the immune enhancing peptide derived from the spike protein of the MERS coronavirus twice or more, specifically twice, three times, five times, seven times, or ten times. can include

The fusion protein may be one in which the signal peptide, the antigen protein, and the peptide derived from the spike protein of MERS coronavirus are linked in order or in reverse order, and may be operably linked to each other directly or indirectly through a linker.

The linker is not particularly limited thereto as long as it exhibits the activity of the fusion protein, but is specifically arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine , Can be linked using amino acids such as valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan, and can be specifically linked by 1 to 50.

The linker may be cleaved or degraded by various biological or chemical actions such as enzymatic action in vivo or in cells, and upon cleavage or degradation of the linker, the peptides are separated from each other in a desired tissue or cell it could be

The antigen protein may be a peptide derived from the spike protein of SARS-CoV-2, specifically a peptide derived from the receptor binding domain (RBD) of the spike protein.

The peptide derived from the spike protein of SARS-CoV-2 may include the amino acid sequence of SEQ ID NO: 3, and may specifically consist of the amino acid sequence of SEQ ID NO: 3.

The polynucleotide encoding the peptide derived from the spike protein of the SARS-CoV-2 coronavirus may include the nucleotide sequence of SEQ ID NO: 4, and may specifically consist of the nucleotide sequence of SEQ ID NO: 4.

The vaccine may be an mRNA vaccine, and specifically, the antigen included in the vaccine composition and the peptide derived from the spike protein of MERS (Middle East Respiratory Syndrome) coronavirus may be included in mRNA form. In addition, the mRNA may be produced through an in vitro transcription (IVT) process.

The vaccine composition may be in the form of a polypeptide or polynucleotide.

As used herein, the term "polynucleotide" is a polymer material to which nucleotides are bound, and refers to DNA or RNA encoding genetic information.

When the vaccine composition is in the form of a nucleic acid, it may be composed of DNA, RNA or a modified form thereof, for example, cDNA, mRNA or a modified form thereof. If the vaccine composition is DNA or modified DNA, it may be composed of A (Adenine), T (Thymine), G (Guanine), C (Cytosine) and/or modified forms thereof, RNA or modified RNA. In one case, it may be composed of A, U (Uridine), G, C and / or modified forms thereof. For example, the modified DNA or RNA is 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2'-O-dimethyluridine (m4U), 2-thiouridine (s2U) , 2' fluorouridine, pseudouridine, 2'-O-methyluridine (Um), 2' deoxyuridine (2' dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2'-O-methyladenosine (m6A), N6,2'-O-dimethyladenosine (m6Am), N6,N6,2'-O-trimethyladenosine (m62Am), 2'-O-methylcity Dean (Cm), 7-methylguanosine (m7G), 2'-O-methylguanosine (Gm), N2,7-dimethylguanosine (m-2,7G), N2,N2,7-trimethylguanosine (m-2,2,7G) and N1-methyl-pseudouridine may include one or more selected from the group consisting of.

Therefore, when the vaccine composition is in the form of RNA, T of the polynucleotide sequence expressing the vaccine composition herein may be expressed by replacing U, and when the vaccine composition is in the form of modified DNA or modified RNA , In the present specification, A, T, G, C, and U of the polynucleotide sequence expressing the vaccine composition may be substituted with their modified forms.

When the vaccine composition is in the form of a nucleic acid, a 5'-cap, a 5'-untranslated region (UTR), a 3'-untranslated region, and a poly(A) tail It may further include one or more selected from the group consisting of, and may specifically include all of the 5'-cap, 5'-untranslated region, 3'-untranslated region, and poly(A) tail.

The vaccine composition may further include lipid nanoparticles as a drug carrier for effectively delivering the vaccine in vivo and/or into cells, and specifically, 1) an antigenic protein or a polynucleotide encoding the same, and/or 2) A peptide derived from the spike protein of MERS coronavirus or a polynucleotide encoding it may be encapsulated in a lipid nanoparticle.

The vaccine composition may be a pharmaceutical composition, and the vaccine composition may include a pharmaceutically acceptable carrier. The "pharmaceutically acceptable carrier" may refer to a carrier or diluent that does not inhibit the biological activity and properties of the compound to be injected without irritating living organisms. Here, the meaning of "pharmaceutically acceptable" means that the application (prescription) does not have toxicity more than is adaptable without inhibiting the activity of the active ingredient. Any type of carrier that can be used in the vaccine composition may be used as long as it is commonly used in the art and is pharmaceutically acceptable. Non-limiting examples of the carrier include lactose, dextrose, maltodextrin, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, glycerol, ethanol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or Mineral oil etc. are mentioned. These may be used alone or in combination of two or more.

The vaccine composition may use a formal or atypical organic or inorganic polymer as an adjuvant for increasing immunogenicity. Adjuvants are generally known to promote immune responses through chemical and physical binding to antigens. As an adjuvant, an irregular aluminum gel, an oil emulsion, or a double oil emulsion, and an immunosol may be used. In addition, various plant-derived saponins, levamisole, CpG dinucleotides, RNA, DNA, LPS, various types of cytokines, and the like can be used to promote an immune response. The immune composition as described above may be used as a composition for inducing an optimal immune response by combining various adjuvants and immune response promoting additives. In addition, stabilizers, inactivating agents, antibiotics, preservatives, and the like may be used as compositions that may be added to vaccines.

The vaccine composition may be prepared as an oral formulation or parenteral formulation according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. The pharmaceutical composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions according to conventional methods.

When formulating the vaccine composition, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, or surfactants.

When the vaccine composition is prepared as an oral dosage form, a formulation such as powder, granule, tablet, pill, dragee, capsule, liquid, gel, syrup, suspension, wafer, etc. according to a method known in the art together with a suitable carrier can be manufactured with Examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, and wheat starch, cellulose, methylcellulose, ethylcellulose, Celluloses such as sodium carboxymethylcellulose and hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil etc. are mentioned. In the case of formulation, if necessary, diluents and/or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be included.

When the vaccine composition is prepared as a parenteral formulation, it may be formulated in the form of an injection, transdermal administration, nasal inhalation, and suppository along with a suitable carrier according to a method known in the art. In the case of formulation as an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, preferably Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine or sterilization for injection water, isotonic solutions such as 5% dextrose, and the like can be used. When formulated as a transdermal formulation, it may be formulated in the form of ointments, creams, lotions, gels, external solutions, pastas, liniments, air rolls, and the like. In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc., and when formulated into suppositories, the base is Witepsol ( witepsol), tween 61, polyethylene glycols, cacao fat, laurin fat, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, and the like may be used.

The vaccine composition may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease with a reasonable benefit/risk ratio applicable to medical treatment or prevention, and the effective dose level is dependent on the severity of the disease, the activity of the drug, and the patient's Age, weight, health, sex, sensitivity to the drug of the patient, administration time of the composition of the present invention used, route of administration and excretion rate, treatment period, factors including drugs used in combination or simultaneous use with the composition of the present invention used, and others It can be determined according to factors well known in the medical field. The vaccine composition may be administered alone or in combination with components known to exhibit therapeutic effects on microbial infectious diseases such as known viruses and bacteria. It is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors.

The dosage of the vaccine composition can be determined by those skilled in the art in consideration of the purpose of use, the degree of addiction of the disease, the patient's age, weight, sex, history, or the type of substance used as an active ingredient. For example, the vaccine composition of the present invention can be administered at about 0.1 ng to about 1,000 mg/kg, preferably 1 ng to about 100 mg/kg per adult, and the frequency of administration of the composition of the present application is particularly limited thereto. However, it can be administered once a day or divided into several doses. The dosage or frequency of administration is not intended to limit the scope of the present application in any way.

Another aspect is to provide a method for preventing or treating bacterial, viral and/or microbial infections and infectious diseases, comprising administering the vaccine composition to a subject. The same parts as described above are also applied to the method.

The term "individual" used throughout the present specification includes, without limitation, mammals, farmed fish, etc., including mice, livestock, humans, etc., which have or are at risk of developing bacterial, viral and/or microbial infections and infectious diseases. can do.

The subject may be excluding humans.

The vaccine composition may be administered singly or in multiple doses in a pharmaceutically effective amount. At this time, the composition may be formulated and administered in the form of a liquid, powder, aerosol, injection, infusion (intravenous gel), capsule, pill, tablet, suppository or patch.

The route of administration of the vaccine composition for preventing or treating bacterial, viral and/or microbial infections and infectious diseases may be administered through any general route as long as it can reach the target tissue.

The vaccine composition may be administered through intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, intrapulmonary administration, intrarectal administration, etc., as desired. there is. However, oral administration can be administered in an unformulated form, and since the vaccine composition can be denatured or decomposed by gastric acid, the oral composition is formulated to coat the active agent or protect it from degradation in the stomach. It can also be administered orally in the form of a form or an oral patch. In addition, the composition may be administered by any device capable of transporting active substances to target cells.

Another aspect is to provide an immune adjuvant or immune enhancer composition for a vaccine, including a peptide derived from a spike protein of MERS (Middle East Respiratory Syndrome) coronavirus or a polynucleotide encoding the same. The same parts as described above also apply to the composition.

As used herein, the term "vaccine adjuvant" refers to a pharmaceutical or immunological agent administered for the purpose of enhancing the immune response of a vaccine.

The immune adjuvant or immune enhancer composition for vaccine may be a composition for improving/promoting the ability to form neutralizing antibodies, and specifically, it may be a composition for enhancing the ability to form neutralizing antibodies against an antigen due to vaccine administration by enhancing immunity.

The adjuvant composition for vaccine may enhance the vaccine effect against microorganisms such as various bacteria and viruses, for example, Japanese encephalitis virus, Heamophilus influenzae type B (HIB), MERS coronavirus, Zika virus, Pseudomonas aeruginosa, pertussis, Mycobacterium tuberculosis, Bacillus anthracis, HAV (Hepatitis A virus), HBV (Hepatitis B virus), HCV (Hepatitis C virus), HIV (human immunodeficiency virus), HSV (Herpes simplex virus), Neisseria meningitidis, Corynebacterium diphtheria, Bordetella pertussis, Clostridium tetani, HPV (human papilloma virus), varicella virus ( Varicella virus, Enterococci, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Enterobacter, Helicobacter pylori, Malaria, Dengue Viruses, Orientia tsutsugamushi, severe fever with thrombocytopenia syndrome Bunyavirus (SFTS Bunyavirus), severe acute respitaroty syndrome-corona virus (SARS-CoV), SARS-CoV CoV-2), influenza virus, Ebola virus, and pneumococcus, one or more bacteria and viruses Vaccine effect It may be one that can promote, and specifically, it may be one that can enhance the vaccine effect against Corona-19 (SARS-CoV-2).

The vaccine adjuvant composition may additionally contain other adjuvant components, for example, Group 2 elements selected from the group consisting of Mg, Ca, Sr, Ba and Ra or salts thereof; Group 4 elements selected from the group consisting of Ti, Zr, Hf and Rf; salts of aluminum or hydrates thereof; Alternatively, dimethyloctadecylammonium bromide may be additionally included. The salts may be formed with, for example, oxides, peroxides, hydroxides, carbonates, phosphates, pyrophosphates, hydrogenphosphates, dihydrogenphosphates, sulfates or silicates.

Specifically, other adjuvant components that may be additionally included are magnesium hydroxide, magnesium carbonate hydroxide, pentahyddate, titanium dioxide, calcium phosphate, calcium carbonate, barium oxide, barium hydroxide, barium peroxide, and one or more of barium sulfate, calcium sulfate, calcium pyrophosphate, magnesium carbonate, magnesium oxide, aluminum hydroxide, aluminum phosphate, and hydrated aluminum potassium sulfate.

The vaccine adjuvant composition may include a pharmaceutically acceptable carrier.

The composition may be a pharmaceutical composition, a health functional food composition, and/or a quasi-drug composition.

The composition may further include a signal peptide or a polynucleotide encoding the signal peptide. The signal peptide may be a signal peptide derived from Gaussian luciferase or a variant thereof.

The signal peptide may include one or more selected from the group consisting of SEQ ID NOs: 11 to 16, and specifically may include one or more selected from the group consisting of SEQ ID NOs: 11 to 16. More specifically, the signal peptide may include the amino acid sequence of SEQ ID NO: 14.

The polynucleotide encoding the signal peptide may include one or more selected from the group consisting of SEQ ID NOs: 17 to 22, and may specifically consist of one or more selected from the group consisting of 17 to 22. More specifically, the polynucleotide sequence encoding the signal peptide may include the nucleotide sequence of SEQ ID NO: 20.

The signal peptide can improve the expression efficiency and extracellular secretion efficiency of the target protein, thereby enhancing the expression and / or activity of the immune enhancing peptide derived from the spike protein of the MERS coronavirus, thereby increasing the ability of the immune enhancing peptide to form neutralizing antibodies And the immune enhancement efficiency can be further improved.

Another aspect is to provide a peptide for enhancing neutralizing antibody formation ability, including a peptide derived from a spike protein of MERS (Middle East Respiratory Syndrome) coronavirus. The same parts as described above also apply to the peptides.

The peptide for enhancing the ability to form neutralizing antibodies may include the amino acid sequence of SEQ ID NO: 1, specifically 80% or more, specifically 90% or more, more specifically 95% or more, more specifically 98% or more of the sequence % or more, most specifically, an amino acid sequence exhibiting 99% or more homology, and includes without limitation any amino acid sequence of a protein exhibiting substantially the same or equivalent efficacy as the peptide. In addition, as a sequence having homology to the above sequence, if it is an amino acid sequence having the same or corresponding biological activity as the peptide of SEQ ID NO described substantially, the case where some sequences have an amino acid sequence that is deleted, modified, substituted or added is also the present invention It is self-evident that it is included in the category of

As used herein, the term "homology" refers to the degree of similarity of nucleotide sequences or amino acid sequences encoding proteins. When the homology is sufficiently high, the expression product of the corresponding gene may have the same or similar activity. In addition, homology can be expressed as a percentage according to the degree of matching with a given amino acid sequence or nucleotide sequence. As used herein, a homologous sequence having the same or similar activity as a given amino acid sequence or nucleotide sequence is indicated by "% homology". For example, hybridization using standard software, specifically BLAST 2.0, that calculates parameters such as score, identity and similarity, or under defined stringent conditions. It can be confirmed by comparing sequences experimentally, and appropriate hybridization conditions defined are within the scope of the art and are 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 peptide for enhancing the ability to form neutralizing antibodies may further include a signal peptide. The signal peptide may be a signal peptide derived from Gaussian luciferase or a variant thereof.

The signal peptide may include one or more selected from the group consisting of SEQ ID NOs: 11 to 16, and specifically may include one or more selected from the group consisting of SEQ ID NOs: 11 to 16. More specifically, the signal peptide may include the amino acid sequence of SEQ ID NO: 14.

The signal peptide can improve the expression efficiency and extracellular secretion efficiency of the target protein, thereby enhancing the expression and / or activity of the immune enhancing peptide derived from the spike protein of the MERS coronavirus, thereby increasing the ability of the immune enhancing peptide to form neutralizing antibodies And the immune enhancement efficiency can be further improved.

Another aspect is to provide a polynucleotide encoding the peptide for enhancing the ability to form neutralizing antibodies. The same parts as described above also apply to the polynucleotide.

As used herein, the term "polynucleotide" is a polymer material to which nucleotides are bound, and refers to DNA or RNA encoding genetic information.

The polynucleotide may be in the form of mRNA, and specifically, may be produced through an in vitro transcription (IVT) process.

The polynucleotide encoding the peptide for enhancing the ability to form neutralizing antibodies may include the nucleotide sequence of SEQ ID NO: 2, specifically 80% or more, specifically 90% or more, more specifically 95% or more, More specifically, nucleotide sequences exhibiting 98% or more, and most specifically, 99% or more homology, are included without limitation as long as they are nucleotide sequences encoding proteins exhibiting substantially the same or equivalent efficacy as the peptides.

In addition, the polynucleotides encoding the peptides are within a range that does not change the amino acid sequence of the protein expressed from the coding region, considering codons preferred in organisms to express the peptides due to codon degeneracy. Various modifications can be made to the coding region in . Accordingly, the polynucleotide may be included without limitation as long as it is a polynucleotide sequence encoding each of the peptides. In addition, a probe that can be prepared from a known sequence, for example, a sequence that hybridizes under stringent conditions to a sequence complementary to all or part of the polynucleotide sequence and encodes a protein having the activity of the peptide, without limitation can be included

The above "stringent conditions" means conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (eg, J. Sambrook et al., ibid.). For example, genes with high homology, 40% or more, specifically 90% or more, more specifically 95% or more, more specifically 97% or more, particularly specifically 99% or more homology 60 ° C., 1XSSC 0.1% SDS, specifically, 60 ° C., 1XSSC 0.1% SDS, which is a condition for hybridizing with each other and not hybridizing with genes with lower homology, or washing conditions for normal Southern hybridization, more specifically can list conditions for washing once, specifically two to three times, at 68 ° C., at a salt concentration and temperature equivalent to 1XSSC 0.1% SDS.

Hybridization requires that two polynucleotides have complementary sequences, although mismatches between bases are possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Thus, the present application may also include substantially similar polynucleotide sequences as well as isolated polynucleotide fragments 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° C. and using the above-described conditions. In addition, the Tm value may be 60 °C, 63 °C or 65 °C, but is not limited thereto and may be appropriately adjusted by those skilled in the art according to the purpose. Appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotide and the degree of complementarity, parameters well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

The polynucleotide may be provided in the form of an expression vector.

As used herein, the term "expression vector" refers to a recombinant vector that can be introduced into a suitable host cell to express a target protein, and refers to a genetic construct containing essential regulatory elements operably linked to express a gene insert. The term "operably linked" means that a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein of interest are functionally linked so as to perform a general function. Operational linkage with a recombinant vector can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and linkage can be facilitated using enzymes generally known in the art. there is.

An expression vector suitable for the present invention may include a signal sequence for membrane targeting or secretion in addition to expression control elements such as a promoter, initiation codon, stop codon, polyadenylation signal, and enhancer. The initiation codon and 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 the lac, tac, T3 and T7 promoters for prokaryotes, the monkey virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus for eukaryotes. (HIV), e.g., the long terminal repeat (LTR) promoter of HIV, the moloney virus, cytomegalovirus (CMV), Epstein Barr virus (EBV), Rouss sarcoma virus (RSV) promoter, as well as the β- actin promoter, human hemoglobin, human muscle creatine, human metallothionein-derived promoters, and the like, but are not limited thereto.

In addition, the expression vector may include a selectable marker for selecting host cells containing the vector. The selectable marker is for selecting cells transformed with the vector, and markers conferring selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or surface protein expression may be used. Transformed cells can be selected because only cells expressing the selectable marker survive in an environment treated with a selective agent. In addition, when the vector is a replicable expression vector, it may include a replication origin, which is a specific nucleic acid sequence at which replication is initiated.

As a recombinant expression vector for inserting a foreign gene, various types of vectors such as plasmids, viruses, and cosmids can be used. The type of recombinant vector is not particularly limited as long as it functions to express a desired gene and produce a desired protein in various host cells of prokaryotic and eukaryotic cells. A vector capable of producing a large amount of a foreign protein in a similar form to can be used.

To express the peptides of the present invention, various combinations of hosts and vectors can be used. Expression vectors suitable for eukaryotic hosts may include, but are not limited to, expression control sequences derived from SV40, bovine papillomavirus, adenovirus, adenoassociated virus, cytomegalovirus, and retrovirus. Expression vectors that can be used in bacterial hosts include, but are not limited to, pET, pRSET, pBluescript, pGEX2T, pUC vectors, col E1, pCR1, pBR322, pMB9 or derivatives thereof. plasmids, plasmids with a wider host range, such as RP4, phage DNA, which can be exemplified by phage lambda derivatives such as λgt10, λgt11 or NM989, and other DNAs such as M13 and filamentous single-stranded DNA phage Phages and the like may be included. A 2° C. plasmid or a derivative thereof may be used for yeast cells, and pVL941 or the like may be used for insect cells.

The polynucleotide encoding the peptide for enhancing the ability to form neutralizing antibodies may further include a sequence encoding a signal peptide. The signal peptide may be a signal peptide derived from Gaussian luciferase or a variant thereof.

The sequence encoding the signal peptide may include one or more selected from the group consisting of SEQ ID NOs: 17 to 22, and may specifically consist of one or more selected from the group consisting of 17 to 22. More specifically, the sequence encoding the signal peptide may include the nucleotide sequence of SEQ ID NO: 20.

The signal peptide can improve the expression efficiency and extracellular secretion efficiency of the target protein, thereby enhancing the expression and / or activity of the immune enhancing peptide derived from the spike protein of the MERS coronavirus, thereby increasing the ability of the immune enhancing peptide to form neutralizing antibodies And the immune enhancement efficiency can be further improved.

Another aspect is to provide a composition for enhancing the ability to form neutralizing antibodies, including a peptide derived from a spike protein of MERS (Middle East Respiratory Syndrome) coronavirus or a polynucleotide encoding the same. The same parts as described above also apply to the composition.

The composition may include a pharmaceutically acceptable carrier.

The composition may be a pharmaceutical composition, a health functional food composition, and/or a quasi-drug composition.

The composition may further include a signal peptide or a polynucleotide encoding the signal peptide. The signal peptide may be a signal peptide derived from Gaussian luciferase or a variant thereof.

The signal peptide may include one or more selected from the group consisting of SEQ ID NOs: 11 to 16, and specifically may include one or more selected from the group consisting of SEQ ID NOs: 11 to 16. More specifically, the signal peptide may include the amino acid sequence of SEQ ID NO: 14.

The polynucleotide encoding the signal peptide may include one or more selected from the group consisting of SEQ ID NOs: 17 to 22, and may specifically consist of one or more selected from the group consisting of 17 to 22. More specifically, the polynucleotide sequence encoding the signal peptide may include the nucleotide sequence of SEQ ID NO: 20.

The signal peptide can improve the expression efficiency and extracellular secretion efficiency of the target protein, thereby enhancing the expression and / or activity of the immune enhancing peptide derived from the spike protein of the MERS coronavirus, thereby increasing the ability of the immune enhancing peptide to form neutralizing antibodies And the immune enhancement efficiency can be further improved.

Another aspect is to provide a transformant comprising a polynucleotide encoding the peptide for enhancing the ability to form neutralizing antibodies or an expression vector containing the same. The same parts as described above are also applied to the transformant.

The term "transformation" as used herein means, for the purpose of the present invention, by introducing a polynucleotide encoding the peptide or an expression vector containing the polynucleotide into a host cell so that the peptide or the polynucleotide encoding the peptide in the host cell It means to be able to manifest. This is achieved by further inserting the same sequence as the chromosome portion of the host cell on both sides of the polynucleotide to integrate the entire polynucleotide encoding the peptide for enhancing the neutralizing antibody formation ability at a specific position, thereby achieving the expression and secretion of the peptide What can be done is also included. As long as the polynucleotide encoding the transformed peptide can be expressed in the host cell, it includes all of these regardless of whether it is inserted into the chromosome of the host cell or located outside the chromosome. In addition, the nucleic acid molecule may include DNA and RNA in the form of a sequence encoding the peptide of the present invention. The polynucleotide or expression vector may be introduced in any form as long as it can be introduced into a host cell and expressed.

The term "transformant" in the present specification may be a host cell into which the polynucleotide or expression vector may be introduced, and may be a transformant other than human.

Host cells suitable for introduction of the expression cassette or expression vector include Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis Or it may be a prokaryotic cell such as Staphylococcus sp. In addition, fungi such as Aspergillus sp., Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp. or Neuro It may be yeast such as Neurospora crassa, other lower eukaryotic cells, cells of higher eukaryotes such as cells of plants or insects. In addition, it may be a mammalian cell, specifically monkey kidney cells 7 (COS7) cells, NSO cells, SP2/0, Chinese hamster ovary (CHO: chinese hamster ovary) cells, W138, young hamster kidney ( BHK: baby hamster kidney) cells, MDCK, myeloma cell lines, HeLa cells, HuT 78 cells, or HEK293 cells may be used, but are not limited thereto.

The transformation method of the present invention includes any method of introducing a nucleic acid into an organism, cell, tissue or organ, and can be performed by selecting a suitable standard technique according to a host cell known in the art. Specifically, electroporation, protoplast fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, agitation using silicon carbide fibers, agrobacterium-mediated transformation, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, lipofectamine and desiccation/inhibition mediated transformation methods, and the like, but are not limited thereto.

The vaccine composition comprising the antigen of the present invention and the peptide derived from the spike protein of MERS (Middle East Respiratory Syndrome) coronavirus is superior to other vaccines in forming neutralizing antibodies, and thus has the advantage of being able to prepare a vaccine with improved efficacy. there is.

1 is a diagram briefly showing the composition of the SARS-CoV-2 vaccine composition of the present invention.
Figure 2 is a diagram confirming the expression of the mRNA form of the SARS-CoV-2 vaccine composition of the present invention.
3 is a diagram showing the size of lipid nanoparticles into which the SARS-CoV-2 vaccine composition of the present invention is incorporated.
4 is a diagram showing the introduction and expression levels of vaccine compositions encapsulated in lipid nanoparticles into cells.
5 is a diagram showing the general antibody expression level against SARS-CoV-2 of the vaccine composition of the present invention.
6 is a diagram showing the expression level of general antibodies against MERS coronavirus in the vaccine composition of the present invention.
7 is a diagram showing the expression level of neutralizing antibodies against SARS-CoV-2 in the vaccine composition of the present invention.
8 is a diagram showing the construction of an expression cassette composed of various signal peptides and variants thereof.
9 is a view confirming the amount of the target protein expressed and secreted after introduction of the expression cassette into cells by Western blotting.
10 is a view showing the construction of an expression cassette composed of various variants of Gaussian luciferase signal peptide (mutation according to a combination of glycine and alanine).
11 is a diagram confirming the amount of the target protein expressed and secreted after introduction of the expression cassette into cells by Western blotting.
12 is a view showing the construction of an expression cassette composed of various variants (variations according to the number of allarines) of the Gaussian luciferase signal peptide.
13 is a diagram confirming the amount of the target protein expressed and secreted after introduction of the expression cassette into cells by Western blotting.

It will be described in more detail through the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Discovery of peptides capable of improving the ability of vaccines to form neutralizing antibodies

The present invention relates to a peptide capable of enhancing the ability of a vaccine to form neutralizing antibodies and a novel SARS-CoV-2 vaccine composition comprising the same. In order to discover the neutralizing antibody-forming ability enhancing peptide, various candidate peptides were investigated, and among them, peptides derived from the receptor binding domain (RBD) of the beta coronavirus MERS (Middle East Respiratory Syndrome) coronavirus were selected. The selected peptide was named a neutralizing antibody formation enhancing peptide, Pro-X.

Specifically, the RBD of MERS coronavirus binds to DPP4/CD26 (Dipeptidyl peptidase-4/cluster of differentiation 26), and the binding of CD26 to the spike protein of MERS coronavirus promotes virus attachment to host cells and virus-cell fusion. It is known to mediate infection.

Therefore, Pro-X derived from the RBD of the MERS coronavirus combines with DPP4/CD26, which is present in large quantities on the surface of immune cells such as T cells, to induce activation of immune cells such as T cells, thereby improving immune function in vivo. In addition, the experiment was conducted with the expectation that the ability to form neutralizing antibodies against the introduced antigen could be improved.

Example 2: Novel SARS-CoV-2 vaccine design and fabrication

In order to confirm that Pro-X developed in Example 1 can improve the ability to form neutralizing antibodies against various viruses other than MERS, as a representative example, the novel Corona 19, which is more effective than existing vaccines, (COVID-19, SARS-CoV-2) virus vaccines were produced.

First, a novel SARS-CoV-2 vaccine was designed/developed by fusing the RBD of the SARS-CoV-2 spike protein with Pro-X of Example 1 (FIG. 1).

Next, in order to prepare the SARS-CoV-2 vaccine designed above in the form of an mRNA vaccine through in vitro transcription (IVT), the following experiments were performed. Meanwhile, the amino acid sequences of the SARS-CoV-2 RBD protein and Pro-X protein of the vaccine and the base sequences encoding them are shown in Table 1 below.

order order
number Pro-X amino acid EAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTETVQLQLQLQ One base sequence GAA GCC AAA CCG TCC GGG AGT GTG GTA GAA CAA GCG GAG GGG GTA GAA TGC GAC TTC TCT CCG CTT CTG TCA GGC ACC CCG CCG CAA GTC TAT AAC TTC AAG AGA CTG GTA TTC ACG AAT TGC AAT TAC AAC CTC ACG AAA CTG CTG AGT CTT TTC TCA GTG AAT GAC TTT ACC TGT AGC CAA ATA TCT CCA GCG GCA ATA GCA TCT AAT TGT TAC TCC TCT CTC ATC TTG GAT TAC TTC AGC TAT CCG TTG TCT ATG AAA AGC GAT CTT AGC GTA TCC TCA GCG GGA CCT ATT TCT CAA TTC AAt TAT AAG CAG TCA TTT AGC AAT CCA ACA TGC TTG ATA CTG GCC ACC GTA CCC CAC AAT TTG ACC ACT ATT ACG AAA CCT TTG AAG TAT AGC TAC ATT AAT AAA TGC TCT CGG TTG TTG TCA GAC GAT CGG ACC GAG GTT CCA CAA CTG GTC AAT GCT AAC CAA TAC AGC CCG TGC GTG TCT ATT GTT CCG TCT ACG GTG TGG GAG GAC GGG GAC TAC TAT CGA AAG CAG CTT TCT CCA CTG GAA GGC GGT GGA TGG CTT GTt GCC TCC GGT TCT ACC GTT GCC ATG ACG GAG CAG CTC CAG ATG GGA TTT GGG ATC ACC GTA CAG TAT GGC ACC GAT ACG AAT AGT GTC TGT CCT AAA CTG 2 SARS-CoV-2 RBD amino acid PTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATV 3 base sequence CCG ACG GAA TCC ATA GTG CGC TTT CCC AAC ATT ACA AAC CTC TGT CCT TTT GGG GAA GTA TTT AAC GCA ACG AGA TTC GCC AGT GTT TAT GCG TGG AAC CGC AAA AGA ATA TCC AAC TGT GTT GCC GAC TAT AGT GTT CTG TAT AAC TCT GCC TCC TTT AGC ACT TTC AAG TGT TAC GGA GTA AGT CCT ACA AAG TTG AAT GAT TTG TGC TTT ACA AAT GTG TAC GCA GAT TCA TTC GTC ATC AGG GGG GAC GAG GTT CGA CAG ATA GCA CCG GGT CAA ACT GGC AAA ATA GCG GAT TAC AAT TAC AAG TTG CCG GAT GAT TTT ACG GGT TGT GTC ATA GCG TGG AAC TCC AAC AAT TTG GAT TCA AAA GTA GGA GGT AAC TAT AAC TAC CTT TAT CGA CTT TTT CGC AAG AGC AAC CTT AAA CCT TTT GAG AGA GAT ATA AGT ACA GAG ATT TAC CAA GCC GGA AGT ACT CCT TGC AAC GGA GTG GAA GGA TTC AAT TGT TAC TTC CCA TTG CAG TCC TAC GGC TTT CAG CCA ACT AAT GGC GTT GGA TAT CAG CCT TAT CGC GTC GTA GTG TTG AGT TTC GAG TTG TTG CAC GCC CCT GCG ACT GTG 4

Specifically, in order to prepare the RBD alone or RBD-Pro-X fusion vaccine composition of SARS-CoV-2, a sequence encoding the same and a sequence encoding the Gaussian luciferase-derived signal peptide variant (SEQ ID NO: 14) Expression cassettes were constructed (SEQ ID NOs: 5 and 7). In order to obtain a PCR product using the expression cassette as a template, mixing was performed under the conditions shown in Table 2 below, and sequences of SEQ ID NOs: 9 and 10 were used as primer sets. In addition, polymerase was performed using Takara's #R051A.

ingredient Volume DNA template 10 ng 1 μl 10 μM forward primer 1 μl 10 μM reverse primer 1 μl 5x Phusion Buffer 4 μl 10 mM dNTPs 0.4 μl polymer raise 0.2 μl 3'DW 12.4 µl all 20 μl

PCR reaction conditions of the mixed samples were performed as shown in Table 3 below.

step Temperature (℃) hour cycle Initial denaturation 98 2 minutes One Denaturation 98 30 seconds 25 Annealing 60 30 seconds Extension 72 1 minute Final Extension 72 5 minutes One

Next, DNA strands that serve as templates for the IVT reaction from the PCR reaction were identified through agarose gel electrophoresis, purified and separated from the agarose gel using Qiagen's #28706 kit, and NEB's #28706 kit was used as a template. mRNA was synthesized using E2060. The composition of the reaction mixture of IVT is shown in Table 4 below.

ingredient Volume (μl) 2X Premixes 10 T7 Polymer Raise 2 Rnase inhibitor One Pseudo-UTP (10 mM) 2.5 5'-Methyl-CTP (10 mM) 2.5 DNA template (10ng/ul) 2 all 20

After mixing under the same conditions as above, the mixture was reacted at 37 °C for 3 hours. In order to remove the template DNA present in the reacted sample, it was treated with DNase I (#E2060) and reacted at 37 ° C. for 30 minutes to degrade the DNA that was the template.

Next, poly A-tailing was performed on mRNA synthesized under the following conditions using NEB's #E2060.

ingredient Volume (μl) DW 20 IVT reactions 20 10X Poly(A) polymerase reaction buffer 5 Poly(A) polymerase 5 all 50

Next, Qiagen's #74204 kit was used to purify the mRNA produced through the IVT reaction, and the concentration of the final product, mRNA, of the purified mRNA was confirmed through BioTek's Synergy HTX Multi-Mode Reader (Fig. 2). .

Example 3: Lipid nanoparticle encapsulation of mRNA

For intracellular delivery and introduction of the mRNA vaccine prepared in Example 2, it was encapsulated in lipid nanoparticles (LNP) in the following manner.

First, to prepare lipid nanoparticles, ionizable lipids, non-cationic lipids, sterols and pegylated lipids (PEG) were mixed. Next, the mRNA to be encapsulated in the lipid nanoparticles was included in a citric acid buffer solution between pH 3.0 and pH 3.5 at a concentration between N/P ratio of 4 to 20. The lipid mixture and the mRNA solution were mixed at a volume ratio of 1:3 and mixed at a rate of 12.0 ml/min. For the preparation of lipid nanoparticles, NanoAssemblr Ignite (NIN0001) equipment manufactured by Precision Nanosystems was used, and the lipid and mRNA solution mixed through the equipment were ultrafiltered to remove ethanol using Amicon 100kDa membrane (Millipore, UFC810024). went through

Example 4: Confirmation of mRNA concentration and size of mRNA-encapsulated lipid nanoparticles

In order to confirm the mRNA concentration and size of the mRNA-encapsulated lipid nanoparticles prepared in Example 3, the following experiments were performed.

First, a Ribogreen assay Kit (Invitrogen, R11490) was used to measure the mRNA content of the lipid nanoparticles encapsulated with the mRNA prepared in Example 3. The solution provided in the Ribogreen kit is a fluorescent substance that stains nucleic acids, and the encapsulation efficiency in lipid nanoparticles can be measured by measuring the amount of RNA in the solution. In order to stain the mRNA inside the lipid nanoparticle, the lipid nanoparticle was digested with a 2% Triton X-100 solution to expose the mRNA. After mixing the decomposed lipid nanoparticle solution and the Ribogreen solution, as a result of measuring with Excitation 485nm and Emission 530nm using a Microplate reader, it was confirmed that the encapsulation was performed with excellent efficiency (Table 6).

NO Sample ID Encapsulation Efficiency (%) Total mRNA Concentration (ug/mL) Encapsulated mRNA Concentration (ug/mL) One LNPs Only - - - 2 SARS-CoV-2 RBD 98.3 51.1 50.3 3 SARS-CoV-2 RBD_Pro-X 98 52.7 51.7

Next, Zetasizer (Malvern, ZSU5700) equipment was used to measure the size of the lipid nanoparticles encapsulated with mRNA prepared in Example 3. After mixing the lipid nanoparticles and phosphate buffered saline (PBS) at a ratio of 1:100, the mixture (1 mL) was placed in a Polystyrene Cuvette (sarstedt, 67.754) and measured. The measured values are the size and dispersion of the lipid nanoparticles. The degree of dispersion of lipid nanoparticles means exactly the polydispersity index (PDI or PI), which indicates the size distribution between lipid nanoparticles. It was confirmed that the size of the prepared lipid nanoparticles was between 90 and 110 d.nm, and the degree of dispersion was within 0.05 to 0.30 (FIG. 3).

Example 5: Introduction of mRNA-encapsulated lipid nanoparticles into cells and confirmation of expression efficiency

In order to confirm whether the lipid nanoparticles prepared in Example 3 effectively introduced and expressed encapsulated mRNA into cells, the following experiment was performed.

Specifically, HeLa cells were seeded in a 60 mm cell culture dish at a concentration of 2 x 10 ⁵ cells/well, and 24 hours later, 1 μg/ml concentration of ApoE4 (lipid nanoparticles were transferred to cells through LDL receptors) based on the cell culture medium. and then the cells were treated with mRNA-encapsulated lipid nanoparticles. After 24 hours of lipid nanoparticle treatment, the cell culture medium and cells were washed with cold PBS, and then washed with RIPA buffer (50 mM Tris-HCl [pH 8.0], 1% NP-40, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 0.5 % deoxycholate). Cell fluid dissolved in RIPA buffer was collected by centrifugation (15,000 RPM) for 10 minutes at 4 ℃, and 5X Sample buffer (250mM Tris-HCl (pH 6.8), 25% glycerol, 2% in 100 μl of supernatant) SDS, 14.4mM beta-mercaptoethanol, 0.1% bromophenol blue) 25 μl was added. After heating the mixed sample at 100 °C for 5 minutes, the supernatant was recovered by centrifugation (15,000 RPM) at 4 °C for 10 minutes. In order to perform Western blotting, 50 μl of the supernatant was subjected to SDS PAGE Gel electrophoresis. For Western blot analysis, anti-His antibody (Cell Signaling Technology, #12698) and anti-GAPDH antibody (Santacruz, SC-1616) were used, and HRP conjugated anti-rabbit IgG (Santacruz, SC-2357) as secondary antibody , and HRP conjugated anti-mouse IgG (Santacruz, SC-516102) were used. The reacted protein was visualized using a Bio-rad chimidoc imaging system after processing SuperSignal ^TM West Pico PLUS Chemiluminescent Substrate, #34580 from Thermo Scientific??. The amount of visualized protein was analyzed through Quantity One® software provided by Bio-rad.

As a result, it was confirmed that the mRNA encapsulated in the lipid nanoparticles could be effectively introduced into cells and expressed (FIG. 4).

Example 6: Confirmation of antibody and neutralizing antibody formation ability of SARS-CoV-2 mRNA vaccine

In order to confirm the general antibody formation and neutralizing antibody formation efficiency of the SARS-CoV-2 mRNA vaccine containing Pro-X, a neutralizing antibody-forming ability enhancing peptide developed in the present invention, the following experiments were performed.

6-1: SARS-CoV-2 mRNA vaccine administration animal experiment

Lipid nanoparticles encapsulated with the SARS-CoV-2 mRNA vaccine prepared in Example 3 were administered to experimental animals to induce an antibody formation reaction.

Specifically, as the experimental animals, specific pathogen-free (SPF) BALB/c mice (male, 7 weeks old) were used, and the experimental animals were divided into 5 animals per experimental group, with an acclimatization period of 7 days and intramuscular administration once/2 weeks. A total of 2 doses were administered (immunization was performed 2 times at 2-week intervals). Before administration, the administration site (femur) was sterilized with 70% alcohol, and administered to the animal's right thigh using a syringe equipped with a 25-27 gauge needle.

Blood collection was performed through the jugular vein, and at a predetermined time, about 0.2 mL of whole blood was injected into a vacutainer tube containing a clot activator, left at room temperature for about 15-20 minutes to coagulate, and serum It was stored in ice-cold conditions until isolation. After blood collection, the blood was centrifuged at 13,000 rpm for 2 minutes to separate the serum, and immediately stored in ice-cold conditions. .

6-2: Confirmation of general antibody formation of SARS-CoV-2 mRNA vaccine

In order to confirm the level of antibody formed in the serum obtained in Example 5-1, general antibody activity was measured using an enzyme-linked immunosorbent assay (ELISA).

Specifically, the animal serum samples of each experimental group were heat-inactivated by heat treatment at 56 ° C. for 30 minutes before analysis, and the serum of each individual in each experimental group was mixed in the same volume (20ul X 4). Mixed serum samples were diluted by 1/5 times from 1/50 times to 1/2x5 ⁹ using dilution solution (PBS). In addition, antibodies to SARS-CoV-2 were measured using an ELISA Kit (CUSABIO, CSB-EL33241HU) coated with SARS-CoV-2 RBD, and antibodies to MERS coronavirus were measured using MERS RBD (ACRO bio, SPD). -M52H6) Protein-coated plates were used.

As a result of measuring the general antibody level, the vaccine containing only SARS-CoV-2 RBD showed the highest antibody level, and neither vaccine containing SARS-CoV-2-RBD_Pro-X fused with Pro-X It was confirmed that a certain level of antibody was formed (FIG. 5).

Next, as a result of measuring the antibody level against MERS coronavirus, it was confirmed that the vaccine containing SARS-CoV-2-RBD_Pro-X could form antibodies (FIG. 6).

6-3: Confirmation of formation of neutralizing antibodies of SARS-CoV-2 mRNA vaccine

In order to confirm the neutralizing antibody titer level (neutralized 　antibody 　titer), which is the level of antibodies capable of blocking the cell penetration of the virus to SARS-CoV-2 formed in the serum obtained in Example 5-1, a fluorescence reduction neutralization test (focus A reduction neutralization test, FRNT50) was performed.

Specifically, 1.5 x 10 ⁴ cells/well of Vero cells were cultured in a 96-well cell culture plate the day before FRNT was performed. Animal serum samples from each experimental group were heat-inactivated by heat treatment at 56 ° C. for 30 minutes before analysis, and serum samples from each individual in each experimental group were mixed in equal volumes (20ul X 4). The mixed serum samples were diluted using DMEM medium containing 2% FBS, and diluted 2-fold starting from 1:10.

The diluted sample was mixed with infectious virus (300 PFU/well) in the same volume (25 μl each of sample and virus), and then incubated at 37° C. for 30 minutes. After washing the cells cultured the previous day with 200 μl of serum-free DMEM, the cells were treated with 50 μl of the mixture and cultured at 37° C. for 4 hours. After washing with PBS, it was fixed with 10% formalin solution (300 μl). After treatment with 100% methanol, the primary antibody [Anti-SARS-CoV2 NP rabbit mab; Sino Biological (Cat:40143-R001), (1:3000)], and secondary antibodies [Anti-rabbit HRP; Biorad; (1:2000)] were sequentially cultured. Thereafter, the cells were cultured in the dark with 30 μl of KPL TrueBlue Peroxidase Substrate (Seracare (Cat: 5510-0030)) for 30 minutes, and the number of blue viral foci was measured using Immunospot to calculate the neutralizing antibody titer.

As a result of the above experiment, the level of neutralizing antibody titer against SARS-CoV-2 showed a certain level even in the case of the vaccine containing only SARS-CoV-2 RBD, but the SARS-CoV-2- It was confirmed that the vaccine containing RBD_Pro-X showed a significantly superior level (FIG. 7).

Based on the above results, it was confirmed that the SARS-CoV-2 vaccine containing Pro-X can slightly reduce the level of antibody formation, but significantly improve the level of neutralizing antibodies, which are important for the actual virus prevention and treatment effect. Therefore, it can be seen that the ability of various virus vaccines to form neutralizing antibodies can be improved by using Pro-X.

Example 7: Confirmation of expression efficiency according to Gaussian luciferase-derived signal peptide and variant sequences thereof

In order to confirm the effect of the Gaussian luciferase-derived signal peptide variant sequence in the expression cassette used in the above example, the following experiment was performed.

7.1: Confirmation of the expression level and secretion efficiency of the expression cassette containing the Gaussian luciferase-derived signal peptide variant

As a representative example of the target protein, a part of the surface glycoprotein sequence (322nd to 524th amino acid) of SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2, NCBI Reference accession MW110903.1) is used, 5'-UTR derived from CYBA and Romboutsia sp. A 3'-UTR sequence derived from the CE17 chromosome (complete genome sequence ID: CP051144.1) was used. In addition, as a signal peptide, a Gaussian luciferase-derived signal peptide wild type (GL-WT), a variant with G-A (glycine-alanine) added to the wild type (GL-G-A1) (SEQ ID NO: 13) or G-A-A (glycine-alanine) -alanine) was added and a sequence encoding the variant (GL-G-A2) (SEQ ID NO: 14) was added. In addition, as a control, a leucine-rich signal peptide (SP-WT) was used, and the same variants as the Gaussian luciferase-derived signal peptide (SP-G-A1 and SP-G-A2) were used. An expression cassette was designed using the above sequences, and the detailed configuration is shown in FIG. 8 .

Using the expression cassette sequences as templates, mRNA was prepared in the same manner as in the above example and introduced into cells.

As a result of the above experiment, when the signal peptide or its variant used as a control was included in the expression cassette, expression and extracellular secretion of the target protein were hardly observed, but when the Gaussian luciferase signal peptide wild type or variant was included, expression of the target protein The expression level and extracellular secretion level of the target protein were increased, and when GAA was added to the end of the variant, it was confirmed that the expression level and extracellular secretion level of the target protein were better (FIG. 9). In addition, the method for deriving a variant in which GAA is added to the terminal is determined to have no effect on the signal peptide used as a control.

7.2: Confirmation of expression level and secretion efficiency according to Gaussian luciferase-derived signal peptide variant sequence

As a representative example of the target protein, a part of the surface glycoprotein sequence (322nd to 524th amino acid) of SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2, NCBI Reference accession MW110903.1) is used, 5'-UTR and 3'-UTR sequences derived from CYBA were used. In addition, as a signal peptide, a sequence encoding a variant in which one or more G (glycine) was added to the wild type signal peptide derived from Gaussian luciferase or a variant in which G-A-A (glycine-alanine-alanine) was added was added. A detailed configuration is shown in FIG. 10 .

Using the expression cassette sequences as templates, mRNA was prepared in the same manner as in Example 1 and introduced into cells.

As a result of the above experiment, it was confirmed that the expression level and extracellular secretion level of the target protein were increased in the case of the expression cassette to which alanine (A) was added than the expression cassette to which glycine (G) was added to the Gaussian luciferase signal peptide wild type (Fig. 11).

7.3: Confirmation of expression level and secretion efficiency according to the number of alanines of Gaussian luciferase-derived signal peptide variants

As a representative example of the target protein, a part of the surface glycoprotein sequence (322nd to 524th amino acids) of SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2, NCBI Reference accession MW110903.1); Alternatively, the above sequence and Pro-X sequence were used, and 5'-UTR and 3'-UTR sequences derived from CYBA were used. In addition, as a signal peptide, a sequence encoding a variant in which 1 G (glycine) was added to the wild type signal peptide derived from Gaussian luciferase and 2 to 4 A (alanine) was added was added. A detailed configuration is shown in FIG. 12 .

Using the expression cassette sequences as templates, mRNA was prepared in the same manner as in Example 1 and introduced into cells.

As a result of the above experiment, it was confirmed that the extracellular secretion level was excellent in the case of the variant in which 2 to 4 A (alanine) were added to the Gaussian luciferase signal peptide variant in which glycine (G) was added. In the case of the variant, it was confirmed that it was superior to other variants, and it was confirmed that the same was applied even when the type of target protein was different (FIG. 13).

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

<110> RNAGENE Inc. <120> Peptide for enhancing neutralizing antibody-producing ability and vaccine composition comprising the same <130> PN144940KR <150> KR 10-2021-0085774 <151> 2021-06-30 <160> 22 <170> KoPatentIn 3.0 <210> 1 <211> 222 <212> PRT <213> Artificial Sequence <220> <223> Pro-X_AA <400> 1 Glu Ala Lys Pro Ser Gly Ser Val Val Glu Gln Ala Glu Gly Val Glu 1 5 10 15 Cys Asp Phe Ser Pro Leu Leu Ser Gly Thr Pro Pro Gln Val Tyr Asn 20 25 30 Phe Lys Arg Leu Val Phe Thr Asn Cys Asn Tyr Asn Leu Thr Lys Leu 35 40 45 Leu Ser Leu Phe Ser Val Asn Asp Phe Thr Cys Ser Gln Ile Ser Pro 50 55 60 Ala Ala Ile Ala Ser Asn Cys Tyr Ser Ser Leu Ile Leu Asp Tyr Phe 65 70 75 80 Ser Tyr Pro Leu Ser Met Lys Ser Asp Leu Ser Val Ser Ser Ala Gly 85 90 95 Pro Ile Ser Gln Phe Asn Tyr Lys Gln Ser Phe Ser Asn Pro Thr Cys 100 105 110 Leu Ile Leu Ala Thr Val Pro His Asn Leu Thr Thr Ile Thr Lys Pro 115 120 125 Leu Lys Tyr Ser Tyr Ile Asn Lys Cys Ser Arg Leu Leu Ser Asp Asp 130 135 140 Arg Thr Glu Val Pro Gln Leu Val Asn Ala Asn Gln Tyr Ser Pro Cys 145 150 155 160 Val Ser Ile Val Pro Ser Thr Val Trp Glu Asp Gly Asp Tyr Tyr Arg 165 170 175 Lys Gln Leu Ser Pro Leu Glu Gly Gly Gly Trp Leu Val Ala Ser Gly 180 185 190 Ser Thr Val Ala Met Thr Glu Gln Leu Gln Met Gly Phe Gly Ile Thr 195 200 205 Val Gln Tyr Gly Thr Asp Thr Asn Ser Val Cys Pro Lys Leu 210 215 220 <210> 2 <211> 666 <212> DNA <213> Artificial Sequence <220> <223> Pro-X_NT <400> 2 gaagccaaac cgtccgggag tgtggtagaa caagcggagg gggtagaatg cgacttctct 60 ccgcttctgt caggcacccc gccgcaagtc tataacttca agagactggt attcacgaat 120 tgcaattaca acctcacgaa actgctgagt cttttctcag tgaatgactt tacctgtagc 180 caaatatctc cagcggcaat agcatctaat tgttactcct ctctcatctt ggattacttc 240 agctatccgt tgtctatgaa aagcgatctt agcgtatcct cagcgggacc tatttctcaa 300 ttcaattata agcagtcatt tagcaatcca acatgcttga tactggccac cgtaccccac 360 aatttgacca ctattacgaa acctttgaag tatagctaca ttaataaatg ctctcggttg 420 ttgtcagacg atcggaccga ggttccacaa ctggtcaatg ctaaccaata cagcccgtgc 480 gtgtctattg ttccgtctac ggtgtgggag gacggggact actatcgaaa gcagctttct 540 ccactggaag gcggtggatg gcttgttgcc tccggttcta ccgttgccat gacggagcag 600 ctccagatgg gatttgggat caccgtacag tatggcaccg atacgaatag tgtctgtcct 660 aaactg 666 < 210> 3 <211> 203 <212> PRT <213> Artificial Sequence <220> <223> SARS-CoV-2 RBD_AA <400> 3 Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro 1 5 10 15 Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp 20 25 30 Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr 35 40 45 Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr 50 55 60 Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val 65 70 75 80 Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gl y Gln Thr Gly Lys 85 90 95 Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val 100 105 110 Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr 115 120 125 Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu 130 135 140 Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn 145 150 155 160 Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe 165 170 175 Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu 180 185 190 Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val 195 200 <210> 4 <211> 609 <212> DNA <213 > Artificial Sequence <220> <223> SARS-CoV-2 RBD_NT <400> 4 ccgacggaat ccatagtgcg ctttcccaac attacaaacc tctgtccttt tggggaagta 60 tttaacgcaa cgagattcgc cagtgtttat gcgtggaacc gcaaaagaat atccaactgt 120 gttgcc gact atagtgttct gtataactct gcctccttta gcactttcaa gtgttacgga 180 gtaagtccta caaagttgaa tgatttgtgc tttacaaatg tgtacgcaga ttcattcgtc 240 atcagggggg acgaggttcg acagatagca ccgggtcaaa ctggcaaaat agcggattac 300 aattacaagt tgccggatga ttttacgggt tgtgtcatag cgtggaactc caacaatttg 360 gattcaaaag taggaggtaa ctataactac ctttatcgac tttttcgcaa gagcaacctt 420 aaaccttttg agagagatat aagtacagag atttaccaag ccggaagtac tccttgcaac 480 ggagtggaag gattcaattg ttacttccca ttgcagtcct acggctttca gccaactaat 540 ggcgttggat atcagcctta tcgcgtcgta gtgttgagtt tcgagttgtt gcacgcccct 600 gcgactgtg 609 <210> 5 <211> 893 <212> DNA <213> Artificial Sequence <220> <223> RBD-expression cassette_NT <400> 5 agatcttaat acgactcact atagggaaat aagacgcgcc tagcagtgtc ccagccgggt 60 tcgtgtcgcc gaattcgcca ccatgggagt gaaagttctt tttgccctta tttgtattgc 120 tgtggccgag gccggcgccg ccccgacgga atccatagtg cgctttccca acattacaaa 180 cctctgtcct tttggggaag tatttaacgc aacgagattc gccagtgttt atgcgtggaa 240 ccgcaaagt atatccaact gttataagttga ctgttgcctga ct ctgcctcctt 300 tagcactttc aagtgttacg gagtaagtcc tacaaagttg aatgatttgt gctttacaaa 360 tgtgtacgca gattcattcg tcatcagggg ggacgaggtt cgacagatag caccgggtca 420 aactggcaaa atagcggatt acaattacaa gttgccggat gattttacgg gttgtgtcat 480 agcgtggaac tccaacaatt tggattcaaa agtaggaggt aactataact acctttatcg 540 actttttcgc aagagcaacc ttaaaccttt tgagagagat ataagtacag agatttacca 600 agccggaagt actccttgca acggagtgga aggattcaat tgttacttcc cattgcagtc 660 ctacggcttt cagccaacta atggcgttgg atatcagcct tatcgcgtcg tagtgttgag 720 tttcgagttg ttgcacgccc ctgcgactgt ggccctggtt ccaaggggct cttcagctca 780 tcatcaccat catcatcatc accatcactg aggatcccct cgccccggac ctgccctccc 840 gccaggtgca cccacctgca ataaatgcag cgaagccggg aaatattaag ctt 893 <210> 6 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> RBD-expression cassette_AA <400> 6 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu 1 5 10 15 Ala Gly Ala Ala Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr 20 25 30 Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser 35 40 45 Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr 50 55 60 Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly 65 70 75 80 Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala 85 90 95 Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly 100 105 110 Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe 115 120 125 Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val 130 135 140 Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu 145 150 155 160 Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser 165 170 175 Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln 180 185 190 Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg 195 200 205 Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Ala 210 215 220 Leu Val Pro Arg Gly Ser Ser Ala His His His His His His His His 225 230 235 240 His His <210> 7 <211 > 1716 <212> DNA <213> Artificial Sequence <220> <223> RBD-Pro-X -expression cassette_NT <400> 7 agatcttaat acgactcact atagggaaat aagacgcgcc tagcagtgtc ccagccgggt 60 tcgtgtcgcc gaattcgcca ccatgggagt gaaagttctt tttgccctta tttgtattgc 120 tgtggccgag gccggcgccg ccccgacgga atccatagtg cgctttccca acattacaaa 180 cctctgtcct tttggggaag tatttaacgc aacgagattc gccagtgttt atgcgtggaa 240 ccgcaaaaga atatccaact gtgttgccga ctatagtgtt ctgtataact ctgcctcctt 300 tagcactttc aagtgttacg gagtaagtcc tacaaagttg aatgatttgt gctttacaaa 360 tgtgtacgca gattcattcg tcatcagggg ggacgaggtt cgacagatag caccgggtca 420 aactggcaaa atagcggatt acaattacaa gttgccggat gattttacgg gttgtgtcat 480 agcgtggaac tccaacaatt tggattcaaa agtaggaggt aactataact acctttatcg 540 actttttcgc aagagcaacc ttaaaccttt tgagagagat ataagtacag agatttacca 600 agccggaagt actccttgca acggagtgga aggattcaat tgttacttcc cattgcagtc 660 ctacggcttt cagccaacta atggcgttgg atatcagcct tatcgcgtcg tagtgttgag 720 tttcgagttg ttgcacgccc ctgcgactgt gactagtgca gaggcagccg ctaaagaggc 780 tgcagctaaa gaagcggcag ccaaggaagc agctgcaaag gcgttggaag ctgaagctgc 840 agcaaaggag gcagctgcga aggaggctgc tgccaaagaa gccgctgcta aagcagaagc 900 caaaccgtcc gggagtgtgg tagaacaagc ggagggggta gaatgcgact tctctccgct 960 tctgtcaggc accccgccgc aagtctataa cttcaagaga ctggtattca cgaattgcaa 1020 ttacaacctc acgaaactgc tgagtctttt ctcagtgaat gactttacct gtagccaaat 1080 atctccagcg gcaatagcat ctaattgtta ctcctctctc atcttggatt acttcagcta 1140 tccgttgtct atgaaaagcg atcttagcgt atcctcagcg ggacctattt ctcaattcaa 1200 ttataagcag tcatttagca atccaacatg cttgatactg gccaccgtac cccacaattt 1260 gaccactatt acgaaacctt tgaagtatag ctacattaat aaatgctctc ggttgttgtc 1320 agacgatcgg accgaggttc cacaactggt caatgctaac caatacagcc cgt gcgtgtc 1380 tattgttccg tctacggtgt gggaggacgg ggactactat cgaaagcagc tttctccact 1440 ggaaggcggt ggatggcttg ttgcctccgg ttctaccgtt gccatgacgg agcagctcca 1500 gatgggattt gggatcaccg tacagtatgg caccgatacg aatagtgtct gtcctaaact 1560 gtctagagcc ctggttccaa ggggctcttc agctcatcat caccatcatc atcatcacca 1620 tcactgataa ctagggatcc cctcgccccg gacctgccct cccgccaggt gcacccacct 1680 gcaataaatg cagcgaagcc gggaaatatt aagctt 1716 <210> 8 <211> 514 <212> PRT <213> Artificial Sequence <220> <223> RBD-Pro-X -expression cassette_AA <400> 8 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu 1 5 10 15 Ala Gly Ala Ala Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr 20 25 30 Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser 35 40 45 Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr 50 55 60 Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly 65 70 75 80 Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala 85 90 95 Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly 100 105 110 Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe 115 120 125 Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val 130 135 140 Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu 145 150 155 160 Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser 165 170 175 Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln 180 185 190 Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg 195 200 205 Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Thr 210 215 220 Ser Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala 225 230 235 240 Lys Glu Ala Ala Ala Lys Ala Leu Glu Ala Glu Ala Ala Ala Lys Glu 245 250 255 Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala Glu 260 265 270 Ala Lys Pro Ser Gly Ser Val Val Glu Gln Ala Glu Gly Val Glu Cys 275 280 285 Asp Phe Ser Pro Leu Leu Ser Gly Thr Pro Pro Gln Val Tyr Asn Phe 290 295 300 Lys Arg Leu Val Phe Thr Asn Cys Asn Tyr Asn Leu Thr Lys Leu Leu 305 310 315 320 Ser Leu Phe Ser Val Asn Asp Phe Thr Cys Ser Gln Ile Ser Pro Ala 325 330 335 Ala Ile Ala Ser Asn Cys Tyr Ser Ser Leu Ile Leu Asp Tyr Phe Ser 340 345 350 Tyr Pro Leu Ser Met Lys Ser Asp Leu Ser Val Ser Ser Ala Gly Pro 355 360 365 Ile Ser Gln Phe Asn Tyr Lys Gln Ser Phe Ser Asn Pro Thr Cys Leu 370 375 380 Ile Leu Ala Thr Val Pro His Asn Leu Thr Thr Ile Thr Lys Pro Leu 385 390 395 400 Lys Tyr Ser Tyr Ile Asn Lys Cys Ser Arg Leu Leu Ser Asp Asp Arg 405 410 415 Thr Glu Val Pro Gln Leu Val Asn Ala Asn Gln Tyr Ser Pro Cys Val 420 425 430 Ser Ile Val Pro Ser Thr Val Trp Glu Asp Gly Asp Tyr Tyr Arg Lys 435 440 445 Gln Leu Ser Pro Leu Glu Gly Gly Gly Trp Leu Val Ala Ser Gly Ser 450 455 460 Thr Val Ala Met Thr Glu Gln Leu Gln Met Gly Phe Gly Ile Thr Val 465 470 475 480 Gln Tyr Gly Thr Asp Thr Asn Ser Val Cys Pro Lys Leu Ser Arg Ala 485 490 495 Leu Val Pro Arg Gly Ser Ser Ala His His His His His His His His 500 505 510 His His <210> 9 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 9 agatcttaat acgactca ct atagggaaat aaga 34 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 10 aatatttccc ggcttcgctg cattt 25 <210> 11 <211> 17 <212> PRT < 213> Artificial Sequence <220> <223> GL-signal peptide <400> 11 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu 1 5 10 15 Ala <210> 12 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> GL-signal peptide-var1 <400> 12 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu 1 5 10 15 Ala Gly <210> 13 <211 > 19 <212> PRT <213> Artificial Sequence <220> <223> GL-signal peptide-var2 <400> 13 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu 1 5 10 15 Ala Gly Ala <210> 14 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> GL-signal peptide-var3 <400> 14 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu 1 5 10 15 Ala Gly Ala Ala 20 <210> 15 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> GL-signal peptide-var4 <400> 15 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu 1 5 10 15 Ala Gly Ala Ala Ala 20 <210> 16 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> GL-signal peptide-var5 <400> 16 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu 1 5 10 15 Ala Gly Ala Ala Ala Ala 20 <210> 17 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> GL-signal peptide_NA <400> 17 atgggagtga aagttctttt tgcccttatt tgtattgctg tggccgaggc c 51 <210> 18 <211> 54 <212> DNA <213> Artificial Sequence <220 > <223> GL-signal peptide-var1_NA <400> 18 atgggagtga aagttctttt tgcccttatt tgtattgctg tggccgaggc cggc 54 <210> 19 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> GL-signal peptide-var2_NA <400> 19 atgggagtga aagttctttt tgcccttatt tgtattgctg tggccgaggc cggcgcc 57 <210> 20 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> GL-signal peptide-var3_NA <400> 20 atgggagtga aagttctttt tgcccttatt tgtattgctg tggccgaggc cggcgccgcc 60 60 <210> 21 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> GL-signal peptide-var4_NA <400> 21 atgggagtga aagttctttt tgcccttatt tgtattgctg tggccgaggc cggcgccgcc 60 gct 63 <210> 22 <211 > 66 <212> DNA <213> Artificial Sequence <220> <223> GL-signal peptide-var5_NA <400> 22 atgggagtga aagttctttt tgcccttatt tgtattgctg tggccgaggc cggcgccgcc 60gctgca 66

Claims (14)

1) antigen; and
2) MERS (Middle East Respiratory Syndrome) Immune-enhancing peptide derived from the spike protein of coronavirus or a polynucleotide encoding the same
A vaccine composition comprising a. The composition according to claim 1, wherein the immune enhancing peptide is capable of enhancing the ability to form neutralizing antibodies. The composition according to claim 1, wherein the immune enhancing peptide comprises the amino acid sequence of SEQ ID NO: 1. The composition of claim 1, wherein the polynucleotide encoding the immune enhancing peptide comprises the nucleotide sequence of SEQ ID NO: 2. The method according to claim 1, wherein the antigen is selected from the group consisting of peptides, proteins, nucleic acids, sugars, pathogens, attenuated pathogens, inactivated pathogens, viruses, virus-like particles (VLPs), cells and cell fragments Characterized in that, the composition. The method according to claim 1, wherein the antigen is Japanese encephalitis virus (Japanese encephalitis virus) antigen, HIB (Heamophilus influenzae type B) antigen, MERS coronavirus antigen, Zika virus antigen, Pseudomonas aeruginosa (Pseudomonas aeruginosa) antigen, whooping cough Antigen, tuberculosis antigen, anthrax antigen, HAV (Hepatitis A virus) antigen, HBV (Hepatitis B virus) antigen, HCV (Hepatitis C virus) antigen, HIV (human immunodeficiency virus) antigen, HSV (Herpes simplex virus) antigens of Neisseria meningitidis, antigens of Corynebacterium diphtheria, antigens of Bordetella pertussis, antigens of Clostridium tetani, HPV (human papilloma virus) antigen, Varicella virus, Enterococci antigen, Staphylococcus aureus antigen, Klebsiella pneumonia antigen, Acinetobacter baumani (Acinetobacter) baumannii antigen, Enterobacter antigen, Helicobacter pylori antigen, malaria antigen, dengue virus antigen, Orientia tsutsugamushi antigen, severe fever with thrombocytopenia syndrome Bunyavirus; SFTS Bunyavirus antigen, severe acute respitaroty syndrome-corona virus; SARS-CoV antigen, SARS-CoV-2 A vaccine composition, characterized in that it is selected from the group consisting of the antigen of 2), an influenza virus antigen, an Ebola virus antigen, and a pneumococcal antigen. The composition according to claim 1, characterized in that the vaccine is in the form of a killed vaccine, an attenuated vaccine, a subunit vaccine, a conjugate vaccine, a recombinant vaccine, a monovalent vaccine, a multivalent vaccine or a combination vaccine. The composition according to claim 1, wherein the composition further comprises a signal peptide or a polynucleotide encoding the signal peptide. The composition according to claim 8, wherein the signal peptide comprises one or more of the amino acid sequences of SEQ ID NOs: 11 to 16. The composition of claim 1, wherein the vaccine composition comprises a fusion protein in which the antigen protein and the peptide are linked or a polynucleotide encoding the same. A vaccine adjuvant composition comprising a peptide derived from a spike protein of MERS (Middle East Respiratory Syndrome) coronavirus or a polynucleotide encoding the same. An immune enhancer or adjuvant composition comprising a peptide derived from a spike protein of MERS (Middle East Respiratory Syndrome) coronavirus or a polynucleotide encoding the same. A peptide for enhancing neutralizing antibody formation, including a peptide derived from a spike protein of MERS (Middle East Respiratory Syndrome) coronavirus. A polynucleotide comprising a polynucleotide encoding the peptide for enhancing the ability to form neutralizing antibodies of claim 13.

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