Motif sequence for enhancing long-term and adaptive immunity, and vaccine composition comprising same

Abstract

The present invention relates to a novel immune-enhancing protein capable of enhancing antibody-forming ability, and use thereof. The vaccine composition comprising an antigen and a gPA-1-derived protein of Pneumocystis carinii or a gpl20-derived protein of human immunodeficiency virus (HIV), of the present invention, has an antibody-forming ability against a disease antigen that is superior to that of other vaccines, and thus a more effective vaccine can be prepared.

Description

Motif sequence for enhancing long-term and adaptive immunity and vaccine composition containing the same

[0001] The present invention relates to a novel immune enhancing protein capable of enhancing antibody formation ability and its use.

[0002] A vaccine is an antigen used to actively immunize animals to prevent infectious diseases, or a biological product containing an antigen as an active ingredient. It was first proposed by the French microbiologist L. Pasteur. In particular, a vaccine as a biological product refers to an immunogen that is administered to animals for the purpose of preventing infectious diseases and induces immunity in the body. Typically, when a vaccine is administered, the body produces the corresponding antibodies and acquires immunity. Once produced, the antibodies remain in the body for a relatively long time, so that even if infection by the pathogen causing the disease occurs, defense is possible, ultimately preventing the disease.

[0003] Typically, there are various types of vaccines, including killed vaccines that use bacteria to kill them, inactivated vaccines, live vaccines that use live bacteria as they are, attenuated vaccines that use attenuated strains of live bacteria, bacterial toxoids or their derivatives, etc. In order to develop an effective vaccine, it is necessary to smoothly form antibodies against the pathogen that causes the disease so that an immune response in the body can be appropriately induced, and therefore it is desirable to develop a vaccine in a form that is as similar to the pathogen that causes the disease as possible.

[0004] Meanwhile, adjuvants play an important role in the prevention and treatment of infectious diseases by activating immune cells. In general, adjuvants are a general term for substances and methods that regulate (strengthen, suppress) immune responses, and refer to methods for administering antigens and substances that act on immune cells to enhance or suppress immune responses antigen-specifically or non-specifically. Known substances with regulatory activity centered on strengthening immune responses include bacterial cells such as BCG and Propionibacterium acnes, cell walls, bacterial components such as trehalose dimycolate (TDM), lipopolysaccharide (LPS) and lipid A fractions, endotoxins of Gram-negative bacteria, β-glucan (polysaccharide), N-acetylmuramyldipeptide (MDP), bestatin, levamisole, and synthetic compounds such as thymus hormones, thymus factors, and taftsin, as well as proteins and peptide substances derived from biological components.

[0005] In addition, lymphokines, monokines, interferons, and cyclophosphamide, which have regulatory activities such as enhancing the immune response by removing suppressive T cells and suppressive macrophages, are also considered as immunoadjuvants in a broad sense. In addition, Freund's incomplete adjuvant (FIA), which is formulated as a water-in-oil emulsion by mixing equal amounts of mineral oil containing Arlacel A and an antigen solution, is used as an immunoadjuvant for antigen administration forms.

[0006] These immune-adjuvant components are known to have effects of not only enhancing but also suppressing immune responses depending on the route of administration, dosage, and timing of administration, and also to have differences in the production of blood antibodies against antigens, induction of cellular immunity, and class of immunoglobulins depending on the type of adjuvant.

[0007] Against this backdrop, the present inventors developed an immune enhancing protein derived from a bacterial and viral glycoprotein capable of enhancing antibody formation ability and a vaccine composition comprising the same, and verified the excellent antibody formation ability of the vaccine, thereby completing the present invention.

[0008] One aspect provides a vaccine composition comprising: 1) an antigen; and 2) an immune enhancing protein derived from a bacterial or viral glycoprotein or a polynucleotide encoding the same.

[0009] 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.

[0010] Another aspect provides an immune adjuvant or immune enhancing composition comprising a protein derived from a bacterial or viral glycoprotein or a polynucleotide encoding the same.

[0011] Another aspect provides a polypeptide for enhancing antibody formation ability, comprising a protein derived from a glycoprotein of bacteria and viruses.

[0012] Another aspect provides a polynucleotide encoding a polypeptide for enhancing antibody formation ability of the present invention.

[0013] One aspect is to provide a vaccine composition comprising 1) an antigen; and 2) an immune enhancing protein derived from a bacterial or viral glycoprotein or a polynucleotide encoding the same.

[0014] The term "vaccine" in this specification refers to a biological preparation containing an antigen that provides immunity to a living organism, and refers to an immunogen or antigenic substance that creates immunity in a living organism by administering it to a human or animal to prevent infection and / or infectious disease.

[0015] The above 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 combined vaccine.

[0016] The immune enhancing protein derived from the glycoprotein of the above bacteria and viruses or the polynucleotide encoding it may include at least one selected from the group consisting of a protein derived from glycoprotein A-1 (gPA-1) of Pneumocystis carinii or a polynucleotide encoding it and a protein derived from gp120 (Envelope glycoprotein GP120) of HIV (Human Immunodeficiency virus) or a polynucleotide encoding it, and specifically, may include a protein derived from gp120 of HIV or a polynucleotide encoding it.

[0017] The gPA-1-derived protein of the above Pneumocystis carinii may include the amino acid sequence of SEQ ID NO: 1, and may be specifically composed of the amino acids of SEQ ID NO: 1. In addition, a sequence that exhibits a homology of 80% or more, specifically 90% or more, more specifically 95% or more, even more specifically 98% or more, and most specifically 99% or more with the above sequence, and a sequence that is substantially identical or exhibits a corresponding effect, is included without limitation.

[0018] The polynucleotide encoding the gPA-1 derived protein of the above Pneumocystis carinii may include the base sequence of SEQ ID NO: 2, and specifically may be composed of the base sequence of SEQ ID NO: 2. In addition, a sequence that exhibits a homology of 80% or more, specifically 90% or more, more specifically 95% or more, even more specifically 98% or more, and most specifically 99% or more with the above sequence, and a sequence that is substantially identical or exhibits a corresponding effect, is included without limitation.

[0019] The above HIV gp120-derived protein may include the amino acid sequence of SEQ ID NO: 3, and specifically may be composed of the amino acid sequence of SEQ ID NO: 3. In addition, a sequence that exhibits a homology of 80% or more, specifically 90% or more, more specifically 95% or more, even more specifically 98% or more, and most specifically 99% or more with the above sequence, and is substantially identical or exhibits a corresponding effect, is included without limitation.

[0020] The polynucleotide encoding the gp120-derived protein of the above HIV may include the base sequence of SEQ ID NO: 4, and specifically may be composed of a sequence having the base sequence of SEQ ID NO: 4. In addition, a sequence that exhibits a homology of 80% or more, specifically 90% or more, more specifically 95% or more, even more specifically 98% or more, and most specifically 99% or more with the above sequence, and a sequence that is substantially identical or exhibits a corresponding effect, is included without limitation.

[0021] The term "homology" as used herein refers to the degree of similarity between a base sequence or amino acid sequence encoding a protein, and if the homology is sufficiently high, the expression product of the corresponding gene may have the same or similar activity. In addition, homology may be expressed as a percentage according to the degree of matching with a given amino acid sequence or base sequence. In this specification, a homologous sequence having the same or similar activity as a given amino acid sequence or nucleotide sequence is expressed as "% homology." For example, it can be confirmed by using standard software that calculates parameters such as score, identity, and similarity, specifically BLAST 2.0, or by comparing sequences through a hybridization experiment performed under defined stringent conditions, and the defined appropriate hybridization conditions are within the scope of the relevant technology and can be determined by a method well known to those skilled in the art.

[0022] Additionally, the gPA-1 derived protein of Pneumocystis carinii or the gp120 derived protein of HIV may have conservative substitutions in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, but is not limited thereto.

[0023] The term "conservative substitution" as used herein refers to the replacement 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 may generally be based on similarities in the 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. Amino acids can also be classified into those with electrically charged side chains and those with uncharged side chains. Charged amino acids include aspartic acid, glutamic acid, lysine, arginine, and histidine, while uncharged amino acids can be further classified as nonpolar or polar amino acids. Nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, and proline; polar amino acids can 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.

[0024] The above-described immune enhancing protein or a polynucleotide encoding the same, and a vaccine composition comprising the same, can enhance the formation ability of antibodies formed by an antigen, and the antibodies may include neutralizing antibodies and / or binding antibodies. Specifically, the immune enhancing protein derived from the glycoprotein of the bacteria or virus or the polynucleotide encoding the same can enhance the formation ability / production ability of binding antibodies and / or neutralizing antibodies.

[0025] The term "neutralizing antibody" as used herein refers to an antibody that neutralizes the biological effects of pathogens or infectious particles when they invade the body, thereby protecting cells. Neutralizing antibodies are part of the adaptive immune system's immune response against viruses, intracellular bacteria, and microbial toxins. Neutralizing antibodies are produced in a form specific to the surface structure of infectious particles and bind to them, preventing the infectious antigen from interacting with host cells and achieving immunity. This reaction is called antibody neutralization. Generally, when a vaccine is administered, the body produces both general antibodies and neutralizing antibodies. General antibodies induce a general immune response, while neutralizing antibodies induce an immune response against a specific antigen. Therefore, a vaccine with a higher number of neutralizing antibodies than general antibodies is considered effective.

[0026] The ability to form / produce the above-mentioned binding antibody and / or neutralizing antibody may be enhanced or improved by enhancing the antibody formation ability by binding an immune enhancing protein derived from a glycoprotein of the above-mentioned bacteria and viruses or a polynucleotide encoding the same as a dendritic cell receptor ligand to an immune cell receptor in the body to enhance the immune function in the body.

[0027] In one embodiment, the immune enhancing protein or a vaccine composition comprising the same may enhance or improve antibody formation and / or production ability against influenza A virus and SARS-COV-2. Specifically, the influenza A virus may include at least one selected from the group consisting of Hawaii / 70 / 2019, California / 04 / 2009, and Wisconsin / 588 / 2019. In addition, the SARS-COV-2 may include at least one selected from the group consisting of BA.4, BA.5, wild type (WT), Delta plus, BA.1, and XBB.1.5.

[0028] The immune enhancing protein derived from the glycoprotein of the above bacteria and viruses or the polynucleotide encoding the same may be used as an immune enhancing agent or an immune adjuvant.

[0029] The term "adjuvant" as used herein refers to a substrate or additive that cannot, on its own, induce specific immunity against the antigen / immunogen of a vaccine, but that, when combined with the antigen, stimulates the immune system and enhances the immune response. In other words, a vaccine utilizing both an antigen and an adjuvant induces a more potent immune response than the antigen alone.

[0030] The vaccine may be an mRNA vaccine, and specifically, the antigens and immune-enhancing proteins derived from bacterial and viral glycoproteins contained in the vaccine composition may be included in the form of mRNA encoding them. Furthermore, the mRNA may be produced through an in vitro transcription (IVT) process.

[0031] The above vaccine composition may be in the form of a polypeptide or polynucleotide.

[0032] The term "polynucleotide" in this specification refers to a polymeric substance in which nucleotides are bound, such as DNA or RNA, which encodes genetic information.

[0033] If 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 a modified DNA, it may be composed of A (Adenine), T (Thymine), G (Guanine), C (Cytosine), and / or a modified form thereof, and if it is RNA or a modified RNA, it may be composed of A, U (Uridine), G, C, and / or a modified form thereof. For example, the modified DNA or RNA may be 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-methylcytidine (Cm), 7-methylguanosine (m7G), 2'-O-methylguanosine (Gm), It may include at least one selected from the group consisting of N2,7-dimethylguanosine (m-2,7G), N2,N2,7-trimethylguanosine (m-2,2,7G) and N1-methyl-pseudouridine.

[0034] Accordingly, when the vaccine composition is in the form of RNA, T of the polynucleotide sequence expressing the vaccine composition in this specification may be expressed by being replaced with U, and when the vaccine composition is in the form of modified DNA or modified RNA, A, T, G, C and U of the polynucleotide sequence expressing the vaccine composition in this specification may be expressed by being replaced with their modified forms, respectively.

[0035] When the above vaccine composition is in the form of a nucleic acid, it may additionally include at least one selected from the group consisting of a 5'-cap, a 5'-untranslated region (UTR), a 3'-untranslated region, and a poly(A) tail, and specifically, it may include all of the 5'-cap, the 5'-untranslated region, the 3'-untranslated region, and the poly(A) tail.

[0036] The above vaccine composition may further include lipid nanoparticles as drug delivery vehicles for effectively delivering the vaccine into a living body and / or into cells, and specifically, 1) an antigen protein or a polynucleotide encoding the same, and / or 2) an immune enhancing protein derived from a bacterial or viral glycoprotein or a polynucleotide encoding the same may be encapsulated in the lipid nanoparticles.

[0037] The above vaccine composition may comprise a fusion protein in which the antigen protein and the immune enhancing protein derived from the glycoprotein of the bacteria and virus are linked, or a polynucleotide encoding the fusion protein.

[0038] The fusion protein may comprise the antigen protein and the immune enhancing protein derived from the glycoprotein of the bacteria and virus more than twice, specifically two, three, five, seven or ten times.

[0039] The above fusion protein may be a protein in which the antigen protein and the immune enhancing protein derived from the glycoprotein of the bacteria and virus are sequentially or reversely linked, and may be operably linked directly or indirectly via a linker to each other.

[0040] The above linker is not particularly limited to this, as long as it exhibits the activity of the fusion protein, but specifically, amino acids such as arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan can be used for connection, and specifically, 1 to 50 of them can be used for connection.

[0041] The above linker may be cleaved or degraded by various biological or chemical actions, such as enzymatic action, in vivo or within cells, and the peptides may be separated from each other within the desired tissue or cell depending on the cleaved or degraded linker.

[0042] In the above fusion protein, the immune enhancing protein may be directly or indirectly linked to the N-terminus or C-terminus of the antigen protein or peptide, and specifically, the immune enhancing protein may be directly or indirectly linked to the C-terminus of the antigen protein or peptide.

[0043] In one embodiment, the fusion protein may optionally be modified at the N-terminus or C-terminus. Specifically, a protecting group may be bonded to the N-terminus or C-terminus of the fusion protein to obtain better chemical stability, enhanced pharmacological properties (half-life, absorbability, potency, efficacy, etc.), altered specificity (e.g., a broad spectrum of biological activity), etc. The protecting group may be an acetyl group, a fluorenyl methoxy carbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, or polyethylene glycol (PEG), but any component that can modify the fusion protein, particularly enhance the stability of the polypeptide, may be included without limitation.

[0044] In the polynucleotide encoding the above fusion protein, the polynucleotide encoding the immune-enhancing protein may be directly or indirectly linked to the 5'-terminal or 3'-terminal of the polynucleotide encoding the antigen protein or peptide, and specifically, the polynucleotide encoding the immune-enhancing protein may be directly or indirectly linked to the 3'-terminal of the polynucleotide encoding the antigen protein or peptide.

[0045] The term "antigen" in this specification means a substance that induces an immune response in a recipient, and in the present invention, any substance that exhibits such an immune response-inducing effect can be used without limitation.

[0046] 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.

[0047] The above antigens include antigens of Coronavirus, antigens of Japanese encephalitis virus, antigens of Heamophilus influenzae type B (HIB), antigens of MERS coronavirus, antigens of Zika virus, antigens of Pseudomonas aeruginosa, antigens of pertussis, antigens of Mycobacterium tuberculosis, antigens of anthrax, antigens of Hepatitis A virus (HAV), antigens of Hepatitis B virus (HBV), antigens of Hepatitis C virus (HCV), antigens of human immunodeficiency virus (HIV), antigens of Herpes simplex virus (HSV), antigens of Neisseria meningitidis, antigens of Corynebacterium diphtheria, antigens of Bordetella pertussis, antigens of Clostridium tetani, Antigens of human papilloma virus (HPV), Varicella virus, Enterococci, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Enterobacter, Helicobacter pylori, malaria, dengue virus, Orientia tsutsugamushi, severe fever with thrombocytopenia syndrome (SFTS) Bunyavirus, severe acute respitaroty syndrome-corona virus (SARS-CoV)It may include at least one selected from the group consisting of an antigen of SARS-CoV, an antigen of SARS-CoV-2, an antigen of Influenza A virus, an antigen of Influenza B virus, an antigen of Ebola virus, and an antigen of Streptococcus pneumoniae;

[0048] The coronavirus may be selected from the group consisting of alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus, and may be specifically porcine epidemic diarrhea virus (PEDV), canine coronavirus (CCV), feline infectious peritonitis virus (FIPV), bovine coronavirus (BCoV or BCV), avian infectious bronchitis virus (IBV), 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 (MERS-CoV), or a combination thereof. SARS-CoV-2 may be the main causative agent of coronavirus disease-2019 (COVID-19).

[0049] The above antigen protein may include hemagglutinin (HA) of an influenza virus or a protein derived therefrom, and may be a protein in which the transmembrane domain is deleted from the hemagglutinin (HA-non TM).

[0050] The above 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.

[0051] The protein derived from Hemagglutinin (HA) of the above influenza virus may include the amino acid sequence of SEQ ID NO: 5, and specifically may be composed of the amino acid sequence of SEQ ID NO: 5. In addition, a sequence that exhibits a homology of 80% or more, specifically 90% or more, more specifically 95% or more, even more specifically 98% or more, and most specifically 99% or more with the above sequence, and a sequence that is substantially identical or exhibits a corresponding effect, is included without limitation.

[0052] The polynucleotide encoding the Hemagglutinin (HA) derived protein of the above influenza virus may include the base sequence of SEQ ID NO: 6, and specifically may be composed of the base sequence of SEQ ID NO: 6. In addition, a sequence that exhibits a homology of 80% or more, specifically 90% or more, more specifically 95% or more, even more specifically 98% or more, and most specifically 99% or more with the above sequence, and is substantially identical or exhibits a corresponding effect, is included without limitation.

[0053] The protein lacking the transmembrane domain in the Hemagglutinin (HA) of the above influenza virus may include the amino acid sequence of SEQ ID NO: 7, and specifically may be composed of the amino acid sequence of SEQ ID NO: 7. In addition, a sequence that exhibits a homology of 80% or more, specifically 90% or more, more specifically 95% or more, even more specifically 98% or more, and most specifically 99% or more with the above sequence, and a sequence that is substantially identical or exhibits a corresponding effect, is included without limitation.

[0054] The polynucleotide encoding a protein lacking the transmembrane domain in the Hemagglutinin (HA) of the above influenza virus may include the base sequence of SEQ ID NO: 8, and specifically may be composed of the base sequence of SEQ ID NO: 8. In addition, a sequence that exhibits 80% or more, specifically 90% or more, more specifically 95% or more, even more specifically 98% or more, and most specifically 99% or more homology with the above sequence, and is substantially identical or exhibits a corresponding effect, is included without limitation.

[0055] The peptide derived from the spike protein of the SARS-CoV-2 may include the amino acid sequence of SEQ ID NO: 9, and may be specifically composed of the amino acid sequence of SEQ ID NO: 9. In addition, a sequence that exhibits a homology of 80% or more, specifically 90% or more, more specifically 95% or more, even more specifically 98% or more, and most specifically 99% or more with the above sequence, and a sequence that is substantially identical or exhibits a corresponding effect, is included without limitation.

[0056] The polynucleotide encoding the peptide derived from the spike protein of the SARS-CoV-2 coronavirus may include the base sequence of SEQ ID NO: 10, and specifically may be composed of the base sequence of SEQ ID NO: 10. In addition, a sequence that exhibits a homology of 80% or more, specifically 90% or more, more specifically 95% or more, even more specifically 98% or more, and most specifically 99% or more with the above sequence, and a sequence that is substantially identical or exhibits a corresponding effect, is included without limitation.

[0057] Considering the variants having the above-described biological equivalent activity, it is interpreted that an antigen, an immune enhancing protein, a vaccine composition comprising the same, or a polynucleotide molecule encoding the same also includes a sequence showing substantial identity with the sequence described in the sequence number. The substantial identity means a sequence showing at least 61% homology, more preferably 70% homology, even more preferably 80% homology, even more preferably 90% homology, even more preferably 95% homology, and most preferably 98% homology, when the sequence and any other sequence are aligned to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art. Alignment methods for sequence comparison are known in the art.

[0058] The above vaccine composition, the fusion protein, or the immune-enhancing peptide may further comprise a signal peptide. The signal peptide may be a signal peptide derived from Gaussian Luciferase or a variant thereof.

[0059] The term "signal peptide" as used herein refers to a short peptide (5 to 30 amino acids) present at the N-terminus of a newly synthesized protein, which directs the protein to be secreted to a designated location via the secretory pathway. The signal peptide may be used interchangeably with "signal peptide" or "secretory peptide."

[0060] The signal peptide may be directly or indirectly linked to the N-terminus of the vaccine composition, the fusion protein or the immune enhancing peptide.

[0061] The signal peptide may include the amino acid sequence of SEQ ID NO: 11 (MGVKVLFALICIAVAEAGAA), and may be specifically composed of the amino acid sequence of SEQ ID NO: 11.

[0062] The above vaccine may be for preventing or treating an infection or infectious disease caused by the above bacteria and / or virus.

[0063] The term “prevention” in this specification means any act of inhibiting or delaying infection with the bacteria and / or virus and the onset of a disease caused by the infection by administration of the vaccine composition.

[0064] The term "treatment" in this specification means any action that improves or benefits the symptoms of a disease already caused by infection with the bacteria and / or virus due to administration of the vaccine composition.

[0065] The above vaccine may be selected from the group consisting of a coronavirus vaccine, a Japanese encephalitis vaccine, a Hemophilus influenzae type B vaccine, a MERS vaccine, a Zika vaccine, a Pseudomonas aeruginosa vaccine, a cancer vaccine, a tuberculosis vaccine, anthrax vaccine, HAV vaccine, HBV vaccine, HCV vaccine, HIV vaccine, herpes simplex vaccine, meningitis vaccine, diphtheria vaccine, pertussis vaccine, tetanus vaccine, varicella vaccine, multidrug-resistant bacteria vaccine, enterococcus vaccine, staphylococcus vaccine, Klebsiella pneumoniae vaccine, Acinetobacter vaccine, Enterobacter vaccine, Helicobacter vaccine, malaria vaccine, dengue vaccine, tsutsugamushi vaccine, severe fever with thrombocytopenia syndrome vaccine, SARS vaccine, Ebola vaccine, influenza A vaccine, influenza B vaccine, COVID-19 vaccine, and pneumococcal vaccine.

[0066] The vaccine composition may be a pharmaceutical composition, and the vaccine composition may include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" may refer to a carrier or diluent that does not stimulate a living organism and does not inhibit the biological activity and properties of the compound to be injected. Here, "pharmaceutically acceptable" means that the vaccine composition does not inhibit the activity of the active ingredient and does not exhibit toxicity exceeding the adaptability of the subject of application (prescription). Any type of carrier that is commonly used in the art and is pharmaceutically acceptable may be used. Non-limiting examples of the carrier include lactose, dextrose, maltodextrin, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, glycerol, ethanol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. These may be used alone or in combination of two or more.

[0067] The above vaccine composition may use structured or amorphous organic or inorganic polymers as immunoadjuvants to increase immunogenicity. Adjuvants are generally known to promote immune responses through chemical and physical binding to antigens. Examples of immunoadjuvants include amorphous aluminum gels, oil emulsions, double oil emulsions, and immunosols. Furthermore, various plant-derived saponins, levamisole, CpG dinucleotides, RNA, DNA, LPS, and various cytokines may be used to promote immune responses. The above-described immune composition can be used as a composition for optimal immune response induction by combining various adjuvants and immune response-promoting additives. Furthermore, stabilizers, inactivators, antibiotics, preservatives, and the like may be added to the vaccine.

[0068] The above vaccine composition may be prepared as an oral or parenteral formulation, depending on the route of administration, by including a pharmaceutically acceptable carrier in addition to the active ingredient, using a conventional method known in the art. The above pharmaceutical composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, topical preparations, suppositories, or sterile injectable solutions, each using a conventional method.

[0069] When formulating the above vaccine composition, it can be prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, or surfactants.

[0070] When the above vaccine composition is manufactured into an oral dosage form, it can be manufactured into a dosage form such as powder, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers, etc., using a suitable carrier and a method known in the art. At this time, 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; celluloses such as cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyols, and vegetable oils. In case of formulation, the formulation may include diluents and / or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants, as needed.

[0071] When the above vaccine composition is prepared as a parenteral formulation, it can be formulated in the form of injections, transdermal administration, nasal inhalation, and suppositories using a suitable carrier according to a method known in the art. When formulated as an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, and preferably, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection, and isotonic solutions such as 5% dextrose can be used. When formulated as a transdermal formulation, it can be formulated in the form of ointments, creams, lotions, gels, external solutions, pastes, liniments, aerosols, etc. 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, or carbon dioxide, and in the case of formulating it as a suppository, the base can be witepsol, tween 61, polyethylene glycol, cacao butter, laurin butter, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene stearate, sorbitan fatty acid ester, etc.

[0072] The above vaccine composition can be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention, and the effective dosage level can be determined according to factors including the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and the excretion rate, the treatment period, drugs used in combination or simultaneously with the composition of the present invention used, and other factors well known in the medical field. The above vaccine composition can be administered alone or in combination with a component known to exhibit a therapeutic effect against a disease caused by a microbial infection such as a known virus or bacteria. It is important to take all of the above factors into consideration and administer an amount that can achieve the maximum effect with the minimum amount without side effects.

[0073] The dosage of the above vaccine composition can be determined by a person skilled in the art in consideration of the purpose of use, the degree of toxicity of the disease, the patient's age, weight, sex, medical history, or the type of substance used as the 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 not particularly limited thereto, but can be administered once a day or administered in divided doses several times. The dosage or frequency of administration does not limit the scope of the present application in any way.

[0074]

[0075] Another aspect provides 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 principles as described above apply to the method.

[0076] The term "subject" as used throughout this specification may include, without limitation, mammals, including mice, livestock, humans, etc., farmed fish, etc., that are susceptible to or at risk of developing bacterial, viral and / or microbial infections and infectious diseases.

[0077] The above entity may exclude humans.

[0078] In one embodiment, the bacteria, virus and / or microorganism may comprise one or more selected from the group consisting of influenza A virus and SARS-CoV-2.

[0079] The above vaccine composition may be administered in single or 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 (Ringel), capsule, pill, tablet, suppository, or patch.

[0080] The route of administration of the vaccine composition for preventing or treating infection and infectious diseases caused by the above bacteria, viruses and / or microorganisms may be through any common route as long as it can reach the target tissue.

[0081] The above vaccine composition may be administered via intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, transdermal patch, oral, intranasal, intrapulmonary, or rectal routes, depending on the intended purpose. However, when administered orally, the vaccine composition may be administered in an unformulated form, and since the vaccine composition may be denatured or decomposed by gastric acid, the oral composition may be administered orally in a form that coats the active agent or is formulated to protect it from decomposition in the stomach, or in the form of an oral patch. In addition, the composition may be administered by any device that allows the active agent to move to target cells.

[0082]

[0083] Another aspect provides a vaccine use of a composition comprising 1) an antigen; and 2) an immunostimulating protein derived from a bacterial or viral glycoprotein, or a polynucleotide encoding the same. The same portions described above also apply to the above use.

[0084] In one embodiment, the protein (immune enhancing protein) derived from the glycoprotein of the bacteria and viruses or the polypeptide comprising the same may optionally be modified at the N-terminus or C-terminus. Specifically, a protecting group may be bonded to the N-terminus or C-terminus of the protein in order to obtain better chemical stability, enhanced pharmacological properties (half-life, absorbability, potency, efficacy, etc.), altered specificity (e.g., broad biological activity spectrum), etc. The protecting group may be an acetyl group, a fluorenyl methoxy carbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, or polyethylene glycol (PEG), but any component that can modify the protein, particularly enhance the stability of the polypeptide, may be included without limitation.

[0085] The composition may comprise a fusion protein in which the antigen protein and the immune enhancing protein derived from the glycoprotein of the bacteria and virus are linked, or a polynucleotide encoding the fusion protein.

[0086]

[0087] Another aspect provides a vaccine use of a fusion protein comprising the antigen protein and an immune-enhancing protein derived from the bacterial or viral glycoprotein, or a polynucleotide encoding the fusion protein. The same portions described above also apply to the above use.

[0088]

[0089] Another aspect provides an immunoadjuvant or immunopotentiating composition comprising a protein derived from a bacterial or viral glycoprotein or a polynucleotide encoding the same. The same aspects described above also apply to the composition.

[0090] The above-mentioned immune adjuvant or immune enhancing composition may be used for vaccine purposes.

[0091] The term "vaccine adjuvant" as used herein means a pharmaceutical or immunological preparation administered for the purpose of enhancing the immune response to a vaccine.

[0092] The above-mentioned immune adjuvant or immune enhancer composition may be a composition for improving / promoting antibody formation ability, and specifically, may be a composition for enhancing immunity to enhance antibody formation ability against an antigen due to vaccine administration.

[0093] The above-mentioned immune adjuvant composition may enhance the vaccine effect against various microorganisms such as bacteria and viruses, for example, Japanese encephalitis virus, HIB (Heamophilus influenzae type B), MERS coronavirus, Zika virus, Pseudomonas aeruginosa, pertussis, tuberculosis, anthrax, 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, Enterococci, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Enterobacter, Helicobacter pylori, malaria, dengue virus, Orientia tsutsugamushi, severe fever with thrombocytopenia syndrome Bunyavirus (SFTS Bunyavirus), severe acute respitaroty syndrome-corona virus (SARS-CoV).It may be capable of enhancing the vaccine effectiveness against one or more bacteria and viruses selected from the group consisting of SARS-CoV, SARS-CoV-2, Influenza A virus, Influenza B virus, Ebola virus, and Streptococcus pneumoniae, and specifically may be capable of enhancing the vaccine effectiveness against Influenza A virus and / or COVID-19 (SARS-CoV-2).

[0094] The above-described immunoadjuvant composition may additionally contain other immunoadjuvant components, for example, a Group 2 element selected from the group consisting of Mg, Ca, Sr, Ba, and Ra or a salt thereof; a Group 4 element selected from the group consisting of Ti, Zr, Hf, and Rf; a salt of aluminum or a hydrate thereof; or dimethyloctadecylammonium bromide. The salt may be formed with, for example, an oxide, a peroxide, a hydroxide, a carbonate, a phosphate, a pyrophosphate, a hydrogen phosphate, a dihydrogen phosphate, a sulfate, or a silicate.

[0095] Specifically, other immunoadjuvants that may be additionally included may be one or more of magnesium hydroxide, magnesium carbonate hydroxide pentahydrate, titanium dioxide, calcium phosphate, calcium carbonate, barium oxide, barium hydroxide, barium peroxide, barium sulfate, calcium sulfate, calcium pyrophosphate, magnesium carbonate, magnesium oxide, aluminum hydroxide, aluminum phosphate, and hydrated aluminum potassium sulfate.

[0096] The above-described immune adjuvant composition may comprise a pharmaceutically acceptable carrier.

[0097] The above composition may be a pharmaceutical composition, a health functional food composition, and / or an over-the-counter drug composition.

[0098]

[0099] Another aspect provides a method for promoting antibody formation, or for adjuvanting and / or enhancing immunity, comprising administering to a subject a composition comprising a protein derived from a bacterial or viral glycoprotein or a polynucleotide encoding the same. The same principles as described above apply to the method.

[0100] The above method may be for preventing or treating infections and infectious diseases caused by bacteria, viruses and / or microorganisms.

[0101]

[0102] Another aspect provides a composition comprising a protein derived from a bacterial or viral glycoprotein or a polynucleotide encoding the same for use as an antibody-promoting agent, an immunoadjuvant, or an immunostimulator. The same principles described above apply to this use.

[0103]

[0104] Another aspect provides the use of proteins derived from bacterial and viral glycoproteins or polynucleotides encoding them for enhancing antibody formation, as immunoadjuvants, or as immune enhancers. The same principles described above apply to these uses.

[0105]

[0106] Another aspect provides a polypeptide for enhancing antibody formation, comprising a protein (immune enhancing protein) derived from a bacterial or viral glycoprotein. The same aspects described above also apply to the polypeptide.

[0107] The above polypeptide may be for use in immune support or immune enhancement.

[0108] The polypeptide for enhancing the antibody formation ability may include an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and specifically, an amino acid sequence showing 80% or more, specifically 90% or more, more specifically 95% or more, even more specifically 98% or more, and most specifically 99% or more homology with the above sequence, and if it is an amino acid sequence of a protein that exhibits substantially the same or corresponding efficacy as the peptide, it is included without limitation. In addition, if it is an amino acid sequence that has a biological activity substantially the same or corresponding to the peptide of the sequence number described as a sequence having homology with the above sequence, it is obvious that even if it has an amino acid sequence in which a part of the sequence is deleted, modified, substituted, or added, it is also included in the scope of the present invention.

[0109] In one embodiment, the polypeptide may optionally be modified at the N-terminus or C-terminus. Specifically, a protecting group may be bonded to the N-terminus or C-terminus of the polypeptide to obtain better chemical stability, enhanced pharmacological properties (half-life, absorbability, potency, efficacy, etc.), altered specificity (e.g., a broad spectrum of biological activity), etc. The protecting group may be an acetyl group, a fluorenyl methoxy carbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, or polyethylene glycol (PEG), but any component that can modify the polypeptide, particularly enhance the stability of the polypeptide, may be included without limitation.

[0110] The term "stability" in this specification may mean not only in vivo stability, which protects the polypeptide from attack by in vivo protein cleavage enzymes, but also storage stability (e.g., room temperature storage stability).

[0111] Additionally, the polypeptide may additionally include an amino acid sequence manufactured for a specific purpose, such as a targeting sequence, a tag, or a labeled residue.

[0112]

[0113] Another aspect is to provide a polynucleotide encoding the peptide for enhancing the antibody formation ability. The same parts described above also apply to the polynucleotide.

[0114] The above polynucleotide may be in the form of mRNA, and specifically may be produced through an in vitro transcription (IVT) process.

[0115] The polynucleotide encoding the peptide for enhancing the neutralizing antibody formation ability may include a base sequence of SEQ ID NO: 2 or SEQ ID NO: 4, and specifically, a base sequence showing 80% or more, specifically 90% or more, more specifically 95% or more, even more specifically 98% or more, and most specifically 99% or more homology with the above sequence, and if it is a base sequence encoding a protein showing substantially the same or corresponding efficacy as the peptide, it is included without limitation.

[0116] In addition, the polynucleotide encoding the above peptides can have various modifications in the coding region within a range that does not change the amino acid sequence of the protein expressed from the coding region, taking into account the codon preferred in the organism that is to express the peptide due to the degeneracy of the codon. Therefore, the polynucleotide can be included without limitation as long as it is a polynucleotide sequence encoding each peptide. In addition, a probe that can be prepared from a known sequence, for example, a sequence that hybridizes under stringent conditions with a complementary sequence to all or part of the polynucleotide sequence, and encodes a protein having the activity of the peptide, can be included without limitation.

[0117] The above "stringent conditions" refer to conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature (e.g., J. Sambrook et al., supra). For example, conditions in which genes with high homology, for example, genes with a homology of 40% or more, specifically 90% or more, more specifically 95% or more, even more specifically 97% or more, and particularly specifically 99% or more, hybridize and genes with lower homology do not hybridize, or conditions in which washing is performed once, specifically twice or three times, at a salt concentration and temperature corresponding to the washing conditions of a typical Southern hybridization, such as 60°C 1XSSC 0.1% SDS, specifically 60°C 0.1XSSC, 0.1% SDS, and more specifically 68°C 0.1XSSC, 0.1% SDS.

[0118] 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 can hybridize with each other. For example, in DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the present application may also encompass isolated polynucleotide fragments that are complementary in their entirety, as well as substantially similar polynucleotide sequences.

[0119] Specifically, polynucleotides having homology can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C and using the conditions described above. In addition, the Tm value may be, but is not limited to, 60°C, 63°C, or 65°C and can be appropriately adjusted by those skilled in the art according to the purpose. The appropriate stringency for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, and the variables are well known in the art.

[0120] The above polynucleotide may be provided in the form of an expression vector.

[0121] The term "expression vector" as used herein refers to a recombinant vector capable of expressing a target protein when introduced into a suitable host cell, and a genetic construct containing essential regulatory elements operably linked to enable expression of the gene insert. The term "operably linked" means that a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding the target protein are functionally linked to perform a general function. The operably linked vector can be produced using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be easily performed using enzymes generally known in the art.

[0122] Suitable expression vectors of the present invention may include signal sequences for membrane targeting or secretion in addition to expression control elements such as a promoter, initiation codon, termination codon, polyadenylation signal, and enhancer. The initiation and termination codons are generally considered to be part of the nucleotide sequence encoding the immunogenic target protein, and must be functional in a subject when the genetic construct is administered, and must be in frame with the coding sequence. Common promoters can be constitutive or inducible and include, but are not limited to, the lac, tac, T3 and T7 promoters in prokaryotes, and the simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoters, human immunodeficiency virus (HIV), e.g., the long terminal repeat (LTR) promoter of HIV, Moloney virus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), Rous sarcoma virus (RSV) promoters, as well as the β-actin promoter, human hemoglobin, human muscle creatine and human metallothionein promoters in eukaryotes.

[0123] Additionally, the expression vector may include a selectable marker for selecting host cells containing the vector. The selectable marker is used to select cells transformed with the vector, and markers that confer selectable phenotypes such as drug resistance, nutrient requirements, cytotoxic agent resistance, or expression of surface proteins may be used. In an environment treated with a selective agent, only cells expressing the selectable marker survive, allowing the transformed cells to be selected. In addition, if the vector is a replicable expression vector, it may include a replication origin, which is a specific nucleic acid sequence where replication begins.

[0124] Recombinant expression vectors for inserting foreign genes can take many forms, including plasmids, viruses, and cosmids. The type of recombinant vector is not particularly limited, as long as it can express the desired gene and produce the desired protein in various host cells, both prokaryotic and eukaryotic. However, vectors that possess a highly active promoter and strong expression capacity, while also being capable of mass-producing foreign proteins in a form similar to that of the native form, are particularly useful.

[0125] To express the peptides of the present invention, various combinations of hosts and vectors can be utilized. Expression vectors suitable for eukaryotic hosts include, but are not limited to, expression control sequences derived from SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus, and retrovirus. Expression vectors that can be used in bacterial hosts include, but are not limited to, bacterial plasmids obtained from Escherichia coli, including pET, pRSET, pBluescript, pGEX2T, pUC vectors, col E1, pCR1, pBR322, pMB9 or derivatives thereof, plasmids having a wider host range such as RP4, phage DNA such as phage lambda derivatives such as λgt10, λgt11 or NM989, and other DNA phages such as M13 and filamentous single-stranded DNA phages. Yeast cells may be used with the 2°C plasmid or derivatives thereof, and insect cells may be used with the pVL941 or the like.

[0126]

[0127] Another aspect provides a composition for enhancing antibody formation, comprising an immune-enhancing protein derived from a bacterial or viral glycoprotein or a polynucleotide encoding the same. The same aspects described above also apply to the composition.

[0128] The composition may comprise a pharmaceutically acceptable carrier.

[0129] The above composition may be a pharmaceutical composition, a health functional food composition, and / or an over-the-counter drug composition.

[0130]

[0131] Another aspect provides a transformant comprising a polynucleotide encoding the polypeptide for enhancing antibody formation ability or an expression vector comprising the same. The same portions as described above also apply to the transformant.

[0132] As used herein, the term "transformation" refers to, for the purposes of the present invention, introducing a polynucleotide encoding the polypeptide, or an expression vector containing the polynucleotide, into a host cell so that the polypeptide or the polynucleotide encoding it can be expressed in the host cell. This also includes the ability to express and secrete the polypeptide by additionally inserting a sequence identical to a portion of the host cell's chromosome on both sides of the polynucleotide, thereby integrating the polynucleotide encoding the polypeptide for enhancing neutralizing antibody formation ability into a specific location as a whole. The polynucleotide encoding the transformed polypeptide includes all of them, regardless of whether it is integrated into the chromosome of the host cell or located outside the chromosome, as long as it can be expressed in the host cell. In addition, the nucleic acid molecule may include DNA and RNA in the form of a sequence encoding the polypeptide of the present invention. The polynucleotide or expression vector may be introduced in any form as long as it can be introduced into the host cell and expressed.

[0133] The term "transformant" in this specification may be a host cell into which the polynucleotide or expression vector can be introduced, and may be a transformant other than a human.

[0134] A host cell suitable for introduction of the above expression cassette or expression vector may be a prokaryotic cell such as Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus sp. In addition, it may be a fungus such as Aspergillus sp., a yeast such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp. or Neurospora crassa, other lower eukaryotic cells, or a cell of a higher eukaryote such as a plant or insect cell. In addition, it may be a mammalian cell, and specifically, monkey kidney cells 7 (COS7: monkey kidney cells) cells, NSO cells, SP2 / 0, Chinese hamster ovary (CHO: Chinese hamster ovary) cells, W138, baby hamster kidney (BHK: baby hamster kidney) cells, MDCK, myeloma cell lines, HeLa cells, HuT 78 cells, or HEK293 cells may be used, but is not limited thereto.

[0135] The transformation method of the present invention includes any method for introducing a nucleic acid into an organism, cell, tissue, or organ, and can be performed by selecting an appropriate standard technique according to the host cell known in the art. Specifically, the transformation method includes, but is not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, stirring 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.

[0136] The vaccine composition comprising the antigen of the present invention and an immune enhancing protein derived from a glycoprotein of bacteria and viruses has an advantage in that it has superior antibody forming ability compared to other vaccines, and thus can produce a vaccine with improved efficacy.

[0137] Figure 1 is a schematic diagram showing the nucleic acid structure of a vaccine composition comprising the immune enhancing protein of the present invention.

[0138] Figure 2 is a drawing describing grouping information for animal testing of a vaccine composition comprising an immune enhancing protein candidate of the present invention and an influenza antigen.

[0139] FIG. 3 is a diagram illustrating grouping information for animal testing of a vaccine composition comprising an immune enhancing protein candidate of the present invention and a SARS-COV-2 antigen.

[0140] Figures 4 and 5 are diagrams showing the expression levels of an mRNA vaccine containing an immune enhancing protein candidate of the present invention and an influenza antigen.

[0141] Figures 6 and 7 are diagrams showing the expression levels of an mRNA vaccine containing an immune enhancing protein candidate of the present invention and a SARS-COV-2 antigen.

[0142] Figure 8 is a diagram showing the binding antibody titer of an mRNA vaccine containing an immune enhancing protein candidate of the present invention and an influenza antigen to Hawaii / 70 / 2019.

[0143] Figure 9 is a diagram showing binding antibody titers for an mRNA vaccine containing an immune enhancing protein candidate of the present invention and an influenza antigen against California / 04 / 2009.

[0144] Figure 10 is a diagram showing the binding antibody titer of an mRNA vaccine containing an immune enhancing protein candidate of the present invention and an influenza antigen to Wisconsin / 588 / 2019.

[0145] Figure 11 is a diagram showing the binding antibody titer for SARS-COV-2 BA.4 / BA.5 of an mRNA vaccine containing an immune enhancing protein candidate of the present invention and a SARS-COV-2 antigen.

[0146] FIG. 12 is a diagram showing binding antibody titers for SARS-COV-2 WT, Delta plus, BA.1, or XBB.1.5 of an mRNA vaccine comprising a gPA-1-derived protein of Pneumocystis carinii or a gp120-derived protein of HIV as an immune enhancing protein of the present invention.

[0147] Figure 13 is a drawing showing the neutralizing antibody titer as a result of an sVNT assay for SARS-COV-2 WT, Delta plus, BA.1, or BA.4 / BA.5 of an mRNA vaccine comprising a gPA-1-derived protein of Pneumocystis carinii or a gp120-derived protein of HIV as an immune enhancing protein of the present invention.

[0148] The present invention will be described in more detail through the following examples. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0149]

[0150] Example 1: Discovery of an immune-enhancing protein that can enhance the antibody-forming capacity of a vaccine.

[0151] The present invention relates to a protein for enhancing immunity that can improve the antibody formation ability of a vaccine, a composition for enhancing immunity containing the same, and a vaccine composition.

[0152] Accordingly, in order to discover an immune enhancing protein for improving the antibody formation ability, various candidate proteins derived from bacteria and viruses (expected to have the function of a ligand targeting a dendritic cell receptor) were investigated, and among these, a protein derived from glycoprotein A-1 (gPA-1) of Pneumocystis carinii and a protein derived from gp120 (Envelope glycoprotein GP120) of Human Immunodeficiency virus (HIV) were selected as final candidate substances.

[0153] The amino acid sequence and base sequence (which may be a DNA or RNA sequence) of the gPA-1-derived protein and gp120-derived protein selected as the final candidate substances of the above-mentioned immune enhancing protein are described in the table below.

[0154] The strain Pneumocystis carinii gPA-1 carriers User Guide NLTDEKCRKYEEKCLLLEEGDPNNLEEKCVKLRDRCYRQRRQGVAKEILLRALEGKVNNKDECKKRMKEICQGLSEYSDELVFSCFNSD KTCEYLQKNHGDSCKPLELEDKELVEKCQEYLEKCYFYGSSCKDTKCDKVNNKCKGKGIEYEGPKLDFSPVREKPRFPEKIEVENLYKKEEAKG IIVGKPKYKTLRDLALLLIKERNGKDEGEKCKKALEDCESFKHLDYGLEELCGDKDKEDRCKELVEVEDRCTNFKLELYLKGLSTEFEKDKESDYF SWGQVSKLVSMEDCIKFESECFHLERVCTNKIGKACENVRVACYKKGQDRVLNRYFQEGLKGLIGDLELVTENLEKCQKSVVGNYTKLK1염기서열AAT CTC ACC GAC GAA AAG TGT CGA AAG TAC GAG GAA AAG TGC CTT CTG TTG GAA GAG GGA GAC CCA AAC AAC TTG GAG GAG AAA TGC GTT AAA CTT AGG GAT CGA TGC TAC AGA CAA CGA AGG CAG GGA GTC GCC AAA GAG ATC CTG CTC CGA GCC CTC GAA GGA AAA GTC AAT AAT AAG GAC GAG TGT AAG AAA CGG ATG AAA GAA ATT TGT CAA GGC TTG AGT GAA TAC TCA GAC GAA CTG GTG TTC AGT TGT TTT AAC TCT GAC AAA ACG TGC GAA TAC TTG CAG AAA AAT CAT GGG GAC TCA TGT AAA CCT CTC GAA AAA GAA TTG GAA GAT AAA GAA CTC GTG GAA AAA TGT CAG GAG TAT CTG GAA AAA TGT TAC TTC TAT GGT TCA TCT TGC AAA GAT ACA AAA TGT GAT AAG GTT AAT AAT AAA TGCAAG GGG AAA GGG ATT GAG TAC GAG GGG CCT AAG TTG GAT TTT TCA CCT GTT CGC GAA AAA CCC CGG TTT CCG GAG AAG ATC GAA GTT GAG AAC CTC TAT AAA AAG GAA GAG GCT AAG GGG ATT ATT GTT GGC AAG CCG AAA TAT AAA ACG CTG AGA GAC CTT GCG CTT TTG CTT ATT AAG GAG AGG AAC GGC AAG GAC GAA GGC GAA AAA TGC AAG AAG GCT CTC GAA GAT TGT GAA TCC TTT AAG CAC CTT GAC TAC GGG TTG GAA GAA CTG TGC GGG GAC AAG GAC AAG GAG GAC AGG TGT AAA GAG CTG GTC GAA GTT GAG GAC AGA TGT ACC AAC TTC AAA CTT GAG TTG TAT TTG AAG GGA TTG AGC ACT GAG TTT GAG AAA GAC AAA GAA TCT GAC TAC TTT AGT TGG GGA CAA GTA TCC AAG CTC GTA AGC ATG GAG GAC TGC ATC AAG TTC GAA AGT GAA TGC TTC CAC CTC GAG AGG GTC TGC ACA AAC AAG ATC GGT AAA GCG TGC GAG AAC GTA AGA GTA GCC TGC TAT AAG AAG GGT CAG GAT AGG GTC CTT AAC CGA TAT TTT CAA GAG GGG TTG AAA GGT CTG ATT GGG GAT CTC GAA TTG GTA ACA GAG AAT CTT GAA AAG TGT CAA AAA TCA GTC GTG GGA AAC TAT ACT AAA CTG AAG2

[0155]

[0156] <h2 style=";text-align:left;direction:ltr">분류서열서열번호HIV의 gp120 유래 단백질아미노산NVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFY KLDIIPIDNDTTSYKLTSCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSV NFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWNNTLKQIASKLREQFGNNKTIIFKQSSGGDPEIVT HSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQIINMWQKVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSNN3염기서열AAC GTA ACC GAG AAC TTC AAC ATG TGG AAG AAT GAC ATG GTC GAA CAG ATG CAC GAA GAT ATC ATT AGT CTT TGG GAC CAA AGC TTG AAA CCT TGC GTG AAG CTG ACC CCG CTG TGC GTA TCC CTG AAA TGT ACG GAC CTG AAG AAC GAT ACT AAT ACC AAC TCA TCA AGT GGA AGA ATG ATA ATG GAA AAG GGC GAG ATC AAG AAC TGT TCT TTT AAT ATA TCC ACC TCT ATC CGC GGT AAG GTG CAA AAA GAG TAT GCC TTT TTT TAC AAG TTG GAC ATC ATC CCC ATT GAT AAT GAT ACC ACG AGC TAT AAG CTT ACT TCC TGT AAT ACC TCA GTT ATC ACC CAG GCT TGT CCG AAG GTG TCC TTT GAG CCA ATA CCA ATT CAT TAC TGT GCG CCT GCC GGG TTC GCC ATC CTC AAG TGC AAT AAC AAG ACC TTC AACGGC ACC GGG CCG TGT ACT AAT GTT TCA ACC GTG CAG TGT ACA CAC GGG ATA CGA CCT GTC GTT TCA ACG CAG CTC CTC TTG AAT GGA AGC TTG GCG GAG GAA GAA GTT GTA ATC AGA AGC GTT AAC TTT ACA GAC AAT GCA AAG ACC ATC ATT GTC CAA CTT AAC ACC TCC GTC GAA ATC AAC TGT ACA CGG CCT AAC AAT ACA CGG AAA AGG ATA CGG ATA CAG CGC GGG CCC GGA CGC GCT TTT GTG ACT ATT GGC AAG ATT GGG AAC ATG CGC CAA GCG CAT TGC AAC ATA AGT CGA GCG AAA TGG AAC AAC ACT CTG AAG CAG ATT GCG TCC AAG CTC CGC GAG CAG TTT GGT AAT AAT AAG ACT ATC ATT TTT AAA CAA TCC AGT GGA GGG GAC CCT GAA ATA GTG ACG CAT AGT TTC AAC TGC GGC GGG GAG TTT TTC TAC TGC AAC TCT ACC CAG CTG TTT AAT TCC ACG TGG TTT AAT AGT ACA TGG AGC ACC GAA GGG AGT AAC AAC ACA GAG GGT AGT GAC ACT ATA ACC CTG CCA TGC CGA ATA AAA CAA ATC ATC AAC ATG TGG CAG AAG GTC GGA AAG GCA ATG TAC GCC CCG CCA ATC AGC GGT CAG ATC AGA TGC AGC TCC AAT ATA ACA GGT CTC CTT TTG ACA CGC GAT GGA GGA AAC TCT AAC AAT4

[0157]

[0158] Example 2: 다이즈강 방타을 다이스 백신 한국어

[0159] In order to produce a vaccine comprising the immunostimulating protein finally discovered in Example 1 or a polynucleotide encoding the same, the following experiments were performed.

[0160] First, as an example of an antigen for producing a vaccine, the hemagglutinin (HA) of the influenza virus or the receptor binding domain (RBD) of SARS-CoV-2 was used as an antigen, and a recombinant antigen containing candidate immune-enhancing proteins derived from various bacteria and viruses was designed.

[0161] Next, the disease-antigen and immune-enhancing protein candidate was produced in the form of an mRNA vaccine through in vitro transcription (IVT) based on the base sequence encoding the recombinant antigen linked by a linker (Fig. 1).

[0162] Meanwhile, the amino acid sequence and base sequence (which may be a DNA or RNA sequence) of the disease antigen used in the vaccine are described below.

[0163] Influenza HA-full length

[0164] 1) Amino acid sequence (SEQ ID NO: 5)

[0165] DTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTARSWSYIVETSNSDNGTCYPGDFINYEELREQLSSVSSFERFEIFPKTSSWPNHDSDKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLNQTYINDKGKEVLVLWGIHHPPTIAAQESLYQNADAYVFVGTSRYSKKFKPEIATRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFTMERDAGSGIIISDTPVHDCNTTCQTPEGAINTSLPFQNVHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREKIDGVKLESTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICI

[0166] 2) 염기서열 (서열번호 6)

[0167] Gat acc ctg tgc att ggc tat cat gcg aac aac agc acc gat acc gtg gat acc gtg gat acc gtg ctg gaa aaa aac gtg acc gtg acc cat agc gtg aac ctg ctg gaa gat aaa cat aac ggc aaa ctg tgc aaa ctg cgc ggc gtg gcc ccg ctg cat ctg ggc aaa tgc aac att gcg ggc tgg att ctg ggc aac ccg gaa tgc gaa agc ctg agc acc gcg cgc agc tgg agc tat att gtg gaa acc agc aac agc gat aa ggc acc tgc tat ccg ggc gat ttt at aac tat gaa gaa ctg cgc gaa cag ctg agc agc gtg agc ttt gaa cgc ttt gaa att ttt ccg aaa acc agc agc tgg ccg aaa cat gat agc gat aaa ggc gtg acc gcg gcg tgc ccg cat gcg ggc gcg aaa agT ttt tat aaa aac ctg att tgg ctg gtg aaa aaa ggc aac agc tat ccg aaa ctg aac cag acc tat att aac gat aaa ggc aaa gaa gtg ctg gtg ctg tgg ggc att cat cat ccg ccg acc att gcg GCA CAA GAG agc ctg tat cag aac gcg gat gcg tat gtg ttt gtg ggc acc agc cgc tat agc aaa aaa ttt aaa ccg gaa att gcg acc cgc ccg aaa gtg cgc gat cag gaa ggc cgc atg aac tat tat tgg acc ctg gtg gaa ccg ggc gat aaa att acc ttt gaa gcg acc ggc aac ctg gtg gtgccg cgc tat gcg ttt acc atg gaa cgc gat gcg ggc agc ggc att att agc gat acc ccg gtg cat gat tgc aac acc acc tgc cag acc ccg gaa ggc gcg att aac acc agc ctg ccg ttt cag aac gtg cat ccg att acc att ggc aaa tgc ccg aaa tat gtg aaa agc acc aaa ctg cgc ctg gcg acc ggc ctg cgc aac gtg ccg agc att cag agc cgc ggc ctg ttt ggc gcg att gcg ggc ttt at gaa ggc ggc tgg acc ggc att gtg gat ggc tgg tat ggc tat cat cat cag aac gaa cag ggc agc ggc tat gcg gcg gat ctg aaa agc acc cag aac gcg att gat aaa att acc aac aaa gtg aac agc gtg att gaa aaa atg aac acc cag ttt acc gcg gtg ggc aaa gaa ttt aac cat ctg gaa aaa cgc att gaa aac ctg aac aaa aaa gtg gat gat ggc ttt ctg gat att tgg acc tat aac gcg gaa ctg ctg gtg ctg ctg gaa aac gaa cgc acc ctg gat tat cat gat agc aac gtg aaa aac ctg tat gaa aaa gtg cgc aac cag ctg aaa aac aac gcg aaa gaa att ggc aac ggc tgc ttt gaa ttt tat cat aaa tgc gat aac acc tgc atg gaa agc gtg aaa aac ggc acc tat gat tat ccg aaa tat agc gaa gaa gcg aaa ctg aac cgc gaa aaa attgat ggc gtg aaa ctg gaa agc acc cgc att tat cag att ctg gcg att tat agc acc gtg gcg agc agc ctg gtg ctg gtg gtg agc ctg ggc gcg att agc ttt tgg atg tgc agc aac ggc agc ctg cag tgc cgc att tgc att

[0168]

[0169] < 인훈엔자 HA-non TM (막투과도메인 가실)>

[0170] 1) amino acid sequence (서열번호 7)

[0171] DTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTARSSWSYIVETSNSDNGTCYPGDFINYEELREQLSSVSSFERFEIFPKTSSWPNHDS DKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLNQTYINDKGKEVLVLWGIHHPPTIAAQESLYQNADAYVFVGTSRYSKKKFKPEIATRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFT MERDAGSGIIISDTPVHDCNTTCQTPEGAINTSLPFQNVHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVNSVIE KMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREKIDGVKLESTRIYQI

[0172] 2.

[0173] gat acc ctg tgc att ggc tat cat gcg aac aac agc acc gat acc gtg gat acc gtg gat acc gtg ctg gaa aaa aac gtg acc gtg acc cat agc gtg aac ctg ctg gaa gat aaa cat aac ggc aaa ctg tgc aaa ctg cgc ggc gtg gcc ccg ctg cat ctg ggc aaa tgc aac att gcg ggc tgg att ctg ggc aac ccg gaa tgc gaa agc ctg agc acc gcg cgc agc tgg agc tat att gtg gaa acc agc aac agc gat aa ggc acc tgc tat ccg ggc gat ttt at aac tat gaa gaa ctg cgc gaa cag ctg agc agc gtg agc ttt gaa cgc ttt gaa att ttt ccg aaa acc agc agc tgg ccg aaa cat gat agc gat aaa ggc gtg acc gcg gcg tgc ccg cat gcg ggc gcg aaa agt ttt tat aaa aac ctg att tgg ctg gtg aaa aaa ggc aac agc tat ccg aaa ctg aac cag acc tat att aac gat aaa ggc aaa gaa gtg ctg gtg ctg tgg ggc att cat cat ccg ccg acc att gcg GCA CAA GAG agc ctg tat cag aac gcg gat gcg tat gtg ttt gtg ggc acc agc cgc tat agc aaa aaa ttt aaa ccg gaa att gcg acc cgc ccg aaa gtg cgc gat cag gaa ggc cgc atg aac tat tat tgg acc ctg gtg gaa ccg ggc gat aaa att acc ttt gaa gcg acc ggc aac ctg gtg gtgccg cgc tat gcg ttt acc atg gaa cgc gat gcg ggc agc ggc att att agc gat acc ccg gtg cat gat tgc aac acc acc tgc cag acc ccg gaa ggc gcg att aac acc agc ctg ccg ttt cag aac gtg cat ccg att acc att ggc aaa tgc ccg aaa tat gtg aaa agc acc aaa ctg cgc ctg gcg acc ggc ctg cgc aac gtg ccg agc att cag agc cgc ggc ctg ttt ggc gcg att gcg ggc ttt at gaa ggc ggc tgg acc ggc att gtg gat ggc tgg tat ggc tat cat cat cag aac gaa cag ggc agc ggc tat gcg gcg gat ctg aaa agc acc cag aac gcg att gat aaa att acc aac aaa gtg aac agc gtg att gaa aaa atg aac acc cag ttt acc gcg gtg ggc aaa gaa ttt aac cat ctg gaa aaa cgc att gaa aac ctg aac aaa aaa gtg gat gat ggc ttt ctg gat att tgg acc tat aac gcg gaa ctg ctg gtg ctg ctg gaa aac gaa cgc acc ctg gat tat cat gat agc aac gtg aaa aac ctg tat gaa aaa gtg cgc aac cag ctg aaa aac aac gcg aaa gaa att ggc aac ggc tgc ttt gaa ttt tat cat aaa tgc gat aac acc tgc atg gaa agc gtg aaa aac ggc acc tat gat tat ccg aaa tat agc gaa gaa gcg aaa ctg aac cgc gaa aaa attgat ggc gtg aaa ctg gaa agc acc cgc att tat cag att

[0174]

[0175] <SARS-CoV-2 RBD>

[0176] 1) Amino acid sequence (SEQ ID NO: 9)

[0177] CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKL PDDFTGCVIAWNSNKLDSKVSGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGVNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLKGTG

[0178] 2) Base sequence (SEQ ID NO: 10)

[0179] TGC ACC CTG AAA AGC TTT ACC GTG GAA AAA GGC ATT TAT CAG ACC AGC AAC TTT CGC GTC CAG CCG ACG GAA TCC ATA GTG CGC TTT CCC AAC ATT ACA AAC CTC TGT CCT TTT GAC 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 AAC GAG GTT AGC CAG ATA GCA CCG GGT CAA ACT GGC AAC ATA GCG GAT TAC AAT TAC AAG TTG CCG GAT GAT TTT ACG GGT TGT GTC ATA GCG TGG AAC TCC AAC AAG TTG GAT TCA AAA GTA AGC GGT AAC TAT AAC TAC CGT TAT CGA CTT TTT CGC AAG AGC AAC CTT AAA CCT TTT GAG AGA GAT ATA AGT ACA GAG ATT TAC CAA GCC GGA AAT AAG CCT TGC AAC GGA GTG GCA GGA GTC AAT TGT TAC TTC CCA TTG CGA TCC TAC AGC TTT CGG CCA ACT TAT GGC GTT GGA CAT CAG CCT TAT CGC GTC GTA GTG TTG AGT TTC GAG TTG TTG CAC GCC CCT GCG ACT GTG TGT GGC CCG AAA AAG AGC ACA AAT CTG GTT AAG AAC AAG TGT GTG AAC TTC AAC TTC AAC GGC CTC AAG GGA ACT GGT

[0180]

[0181] Next, the mRNA produced above was encapsulated into lipid nanoparticles to produce an mRNA-LNP vaccine formulation. First, four types of lipids, SS-OP, DOPC, cholesterol, and PEG-lipid, were dissolved in 100% ethanol to a concentration of 18.75 mM. These were mixed in a molar ratio of 55:10:33.5:1.5 to prepare a lipid mixture. Next, to encapsulate mRNA into the LNP, IVT mRNA was diluted with DEPC DW to a concentration of 1 ug / ul, and 100 mM sodium citrate (pH 4.0) was added to a final concentration of 50 mM to prepare an mRNA mixture. The lipid mixture:mRNA mixture was mixed in a ratio of 1:3 to produce an mRNA-LNP complex using a microfluidic device from Precision Nanosystems, Canada.

[0182] Next, to confirm the particle characteristics such as the size and mRNA encapsulation efficiency of the mRNA-encapsulated LNPs, 10 μl of each prepared LNP was mixed with 800 μl of PBS (Gibco, #70011-044), transferred to a cuvette (Malvern; Cat # DTS0012), and the particle size and polydispersity index (PDI) were measured using a Zetasizer nanoparticle analyzer from Malvern and the XPLORER program. The program settings were Cell-DTS0012, Material-LNP, Dispersant-PBS, and the number of measurements was adjusted to 3 repetitions. As a result, the LNPs encapsulating IVT mRNA showed a size of 58.3–89.1 nm and a polydispersity index of 0.026–0.068.

[0183] Next, the zeta potential of the LNP was measured. Using a DTS1080 cuvette, the program settings were changed to Cell-DTS1080, and [Advanced setting] - [Measurement process] was changed to manual. As a result, the manufactured LNP was measured to have a zeta potential of -4.88 to 0.147.

[0184] In addition, the mRNA encapsulation efficiency and concentration of the IVT mRNA encapsulated into the LNP during each LNP manufacturing process were measured by performing a Ribogreen assay using the Quanti-iT Ribogreen kit (Invitrogen, # R11490), and the encapsulation efficiency was high, ranging from approximately 90 to 96%.

[0185] Meanwhile, in order to conduct various experiments (cell experiments and animal experiments) on vaccines containing the above disease antigens and various immune-enhancing protein candidates, the control group and experimental group were designated as G1 to G26 as described in FIGS. 2 and 3.

[0186]

[0187] Example 3: Expression Evaluation of a Vaccine Containing an Immunoenhancing Protein

[0188] In order to evaluate whether an mRNA vaccine containing the immune-enhancing protein (or a polynucleotide encoding the same) produced in Example 2 above was expressed, the following experiment was performed.

[0189] Specifically, in order to confirm whether the mRNA-LNP encoding the recombinant protein containing the influenza virus or COVID-19 virus antigen and the immune-enhancing protein produced above can actually produce the protein in cells, the human cell line Huh-7 was transfected with the LNP encapsulating the mRNA produced above. First, Huh-7 cells were cultured for 3 days in RPMI1640 (Welgene, #LM011-03) medium containing 10% heat-inactivated FBS (Gibco, #16000-44) and 1% Penicillin-Streptomycin (Gibco, #15140-122) at 37°C and 5% CO2. After washing with 1x PBS buffer, the cells were detached from the bottom by trypsinization, and the detached cells were seeded in 6-well plates at 2.2 Y 10 5 / well were subcultured. The next day, Opti-MEM (Gibco, #31985-062) and ApoE4 were mixed and incubated at 37°C for 10 minutes, and 1 μg of the prepared mRNA-LNP was mixed and incubated at 37°C for 10 minutes, and then transfected into the cells. 24 hours after transfection, approximately 1 ml of the cell culture medium was transferred to a 1.5 ml tube and the remainder was removed. The cells were washed with cold PBS and collected using RIPA buffer (50 mM Tris-HCl [pH 8.0], 1% NP-40, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 0.5% deoxycholate). The cell solution dissolved in the RIPA buffer was centrifuged (13,000 RPM) at 4°C for 10 minutes, and only the supernatant was recovered. Thermo Scientific TM Pierce of the company TMThe protein concentration in the cell lysate was confirmed using a BCA assay kit. Next, 5X Sample buffer (250 mM Tris-HCl (pH 6.8), 25% glycerol, 2% SDS, 14.4 mM beta-mercaptoethanol, 0.1% bromophenol blue) was added to the cell lysate with the same protein concentration, and the mixed sample was heated at 95°C for 10 minutes to perform Western blotting. 45 μl was then subjected to SDS PAGE gel electrophoresis. Anti-His antibody (Cell Signaling Technology, #12698) and anti-GAPDH antibody (Santacruz, SC-1616) were used for Western blotting analysis, and HRP-conjugated anti-rabbit IgG (Santacruz, SC-2357) and HRP-conjugated anti-mouse IgG (Santacruz, SC-516102) were used as secondary antibodies. The target protein is Thermo Scientific TM SuperSignal of the company TM After treatment with West Pico PLUS Chemiluminescent Substrate, #34580, the samples were analyzed using a Bio-rad chemi-doc imaging system.

[0190] As a result, it was confirmed that the influenza virus or COVID-19 virus antigen introduced into cells using mRNA-LNP was effectively expressed within the cells (Figs. 4 to 7).

[0191]

[0192] Example 4: Evaluation of antibody formation ability of vaccine containing immune enhancing protein

[0193] In order to evaluate the antibody formation ability of the mRNA vaccine containing the immune-enhancing protein (or polynucleotide encoding it) produced in Example 2 above against an antigen, the following experiment was performed.

[0194] First, to evaluate the binding antibody formation ability, each test substance was administered to animals twice with an equal RNA molar number (HA: 8.55 pmol, SARS-CoV-2: 15.5 pmol) at a two-week interval. The binding antibody formation ability was evaluated using serum collected 14 days after the second administration. Specifically, blood was collected by orbital bleed two weeks after the first LNP administration, and by cardiac bleed (whole blood collection) two weeks after the second LNP administration. The collected blood was placed in an anticoagulant-free blood tube (SST container), left at room temperature for more than 30 minutes, and then centrifuged (8,000 rpm, 4℃, 10 minutes) to separate the serum. To measure binding antibody titers against influenza A virus antigen or SARS-COV-2 antigen using the separated serum, an enzyme-linked immunosorbent assay (ELISA) was performed as follows. ELISA was performed using serum collected two weeks (week 4, 4w Serum) after the second administration of the mRNA vaccine candidate.

[0195] 1) Collect a portion of serum from each individual in each group, pool it, heat inactivate it using a water bath at 56℃ for 30 minutes, and centrifuge it at 13,000 rpm for 3 minutes to separate only the supernatant.

[0196] 2) Dilute the protein to be coated (antigen to be analyzed by binding antibody) with PBS as much as necessary to 0.2 ㎍ / well in an immunoplate (SPL, #32396), add 100 ㎕ to each well, attach a plate sealer (Thermo scientific, #236370) well, and incubate at 4℃, overnight (O / N).

[0197] 3) The next day, discard the solution containing the coated antigen protein and wash 5 times with 200 ㎕ of PBS-T (Biosesang, #PR2195-100-00) per well.

[0198] 4) Make a blocking solution containing 1% BSA (GenDepot, #A0100-010) in PBST, add 200 ㎕ to each well, and incubate at room temperature for 2 hours.

[0199] 5) Discard the blocking solution and wash 5 times with 200 ㎕ of PBS-T per well.

[0200] 6) Dilute the serum prepared in step 1 with blocking solution and prepare at different concentrations. Add 100 ㎕ of the diluted serum sample to each well and incubate at room temperature for 2 hours.

[0201] 7) Discard the reaction solution and wash 5 times with 200 ㎕ of PBS-T per well.

[0202] 8) Dilute the secondary antibody m-IgGκ BP-HRP (MyBiosource, #MBS135230) with 1% BSA+PBST to a ratio of 1:4000, add 100 ㎕ to each well, and incubate at room temperature for 1 hour.

[0203] 9) Discard the reaction solution and wash 5 times with 200 ㎕ of PBS-T per well.

[0204] 10) Add 100 ㎕ of TMB solution (Sigma-aldrich, #T4444) to each well and incubate for 1 hour at room temperature, blocking light.

[0205] 11) Add 50 ㎕ of stop solution (Thermo scientific, #N600) to each well, incubate for about 5 minutes at room temperature, and measure the absorbance at 450 nm and 600 nm using a Synergy HTX Multi-Mode Reader (BioTek; #S1LA).

[0206] Next, to evaluate the ability to form neutralizing antibodies, the sVNT assay was performed as follows.

[0207] 1) In separate tubes, diluted positive control, diluted negative control, and collected serum samples were mixed with diluted HRP-RBD solution in a 1:1 volume ratio. For example, 60 μl of positive control was mixed with 60 μl of HRP-RBD solution. The mixture was reacted at 37°C for 30 minutes. Meanwhile, serum samples were diluted 10, 100, and 1000 times with the diluting solution and analyzed.

[0208] 2) Add 100 μl of the positive control mixture, negative control mixture, and sample mixture to the corresponding wells and react at 37°C for 15 minutes.

[0209] 3) Add 260 ㎕ of the washing solution to each well and wash 4 times.

[0210] 4) After the washing step, gently tap the plate on a paper towel to completely remove any solution remaining in the wells.

[0211] 5) Add 100 μl of TMB solution to each well and incubate the plate at 20-25°C for 15 minutes (in the dark).

[0212] 6) Stop the reaction by adding 50 μl of stop solution to each well, and measure the absorbance at 450 nm after 5 minutes using a Synergy HTX Multi-Mode Reader (BioTek; # S1LA).

[0213]

[0214] 4-1: Evaluation of binding antibody formation ability against influenza A virus

[0215] First, as a result of evaluating the antibody formation ability against the influenza A virus Hawaii / 70 / 2019, in the G3 to G12 experimental groups containing the HA of the influenza virus as an antigen, it was confirmed that the vaccine containing a gPA-1-derived protein (G8) or a gp120-derived protein (G9) as an immunostimulatory substance showed a significantly superior binding antibody titer than the vaccine containing proteins derived from other bacteria and viruses, and in particular, it was confirmed that the vaccine containing a gp120-derived protein (G9) showed a significantly superior binding antibody titer (Fig. 8).

[0216] Next, as a result of evaluating the antibody formation ability against the influenza A virus California / 04 / 2009, it was confirmed that the vaccine containing a gp120-derived protein (G9) as an immune-enhancing substance showed significantly superior binding antibody titers than vaccines containing proteins derived from other bacteria and viruses, compared to the G4 experimental group that did not contain a sequence encoding an immune-enhancing protein (Fig. 9).

[0217] Next, as a result of evaluating the antibody formation ability against the influenza A virus Wisconsin / 588 / 2019, it was confirmed that the vaccine containing the gPA-1-derived protein (G8) or gp120-derived protein (G9) as an immune-enhancing substance showed significantly superior binding antibody titers than the vaccine containing proteins derived from other bacteria and viruses, compared to the G4 experimental group that did not contain a sequence encoding an immune-enhancing protein, and in particular, it was confirmed that the vaccine containing the gp120-derived protein (G9) showed significantly superior binding antibody titers (Fig. 10).

[0218]

[0219] 4-2: Evaluation of antibody formation capacity against SARS-CoV-2

[0220] First, as a result of evaluating the antibody formation ability against SARS-CoV-2 BA.4 / BA.5, in the G13 to G26 experimental groups containing the RBD of SARS-CoV-2 as an antigen, compared to the G13 experimental group that did not contain a sequence encoding an immune-enhancing protein, it was confirmed that the vaccine containing a gPA-1-derived protein (G20) or a gp120-derived protein (G23) as an immune-enhancing substance showed a significantly superior binding antibody titer than the vaccine containing proteins derived from other bacteria and viruses, and in particular, it was confirmed that the vaccine containing a gp120-derived protein (G23) showed a significantly superior binding antibody titer (Fig. 11).

[0221] Next, the antibody formation ability against SARS-CoV-2 WT, Delta plus, BA.1 or XBB.1.5 was evaluated, and it was confirmed that the vaccine containing gPA-1-derived protein (G20) or gp120-derived protein (G23) as an immune-enhancing substance showed significantly superior binding antibody titers compared to the G13 experimental group that did not contain a sequence encoding an immune-enhancing protein, and in particular, it was confirmed that the vaccine containing gp120-derived protein (G23) showed significantly superior binding antibody titers (Fig. 12).

[0222] Next, the neutralizing antibody formation ability against SARS-CoV-2 WT, Delta plus, BA.1, or BA.4 / BA.5 was evaluated using the sVNT assay. As a result, it was confirmed that vaccines containing gPA-1-derived protein (G20) or gp120-derived protein (G23) as an immune-enhancing substance showed significantly superior neutralizing antibody titers compared to the G13 experimental group that did not include a sequence encoding an immune-enhancing protein, and in particular, it was confirmed that vaccines containing gp120-derived protein (G23) showed significantly superior neutralizing antibody titers (Fig. 13).

[0223] Based on the above results, it can be seen that the recombinant antigen including the gPA-1-derived protein of Pneumocystis cari and the gp120-derived protein of HIV, which are immune-enhancing proteins discovered in Example 1, exhibit excellent neutralizing antibody and / or binding antibody formation ability, and in particular, it can be seen that the gp120-derived protein has a remarkably excellent effect of enhancing the neutralizing antibody and / or binding antibody formation ability.

[0224]

[0225] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. 1) Antigen; and 2) An immune enhancing protein derived from a bacterial or viral glycoprotein or a polynucleotide encoding the same A vaccine composition comprising:

2. A composition according to claim 1, wherein the immune enhancing protein is capable of enhancing antibody formation ability.

3. A composition according to claim 1, wherein the immune enhancing protein derived from a bacterial or viral glycoprotein or a polynucleotide encoding the same comprises at least one selected from the group consisting of a protein derived from glycoprotein A-1 (gPA-1) of Pneumocystis carinii or a polynucleotide encoding the same and a protein derived from envelope glycoprotein GP120 (gp120) of human immunodeficiency virus (HIV) or a polynucleotide encoding the same.

4. A composition according to claim 3, wherein the gPA-1 derived protein of Pneumocystis carinii comprises an amino acid sequence of sequence number 1.

5. A composition according to claim 3, wherein the polynucleotide encoding the gPA-1 derived protein of Pneumocystis carinii comprises the base sequence of SEQ ID NO:

2.

6. A composition according to claim 3, wherein the HIV gp120-derived protein comprises an amino acid sequence of sequence number 3.

7. A composition according to claim 3, wherein the polynucleotide encoding the HIV gp120-derived protein comprises the base sequence of sequence number 4.

8. A composition according to claim 1, characterized in that 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.

9. In claim 1, the antigen is an antigen of Coronavirus, an antigen of Japanese encephalitis virus, an antigen of HIB (Heamophilus influenzae type B), an antigen of MERS coronavirus, an antigen of Zika virus, an antigen of Pseudomonas aeruginosa, an antigen of pertussis, an antigen of tuberculosis, an antigen of anthrax, an antigen of HAV (Hepatitis A virus), an antigen of HBV (Hepatitis B virus), an antigen of HCV (Hepatitis C virus), an antigen of HIV (human immunodeficiency virus), an antigen of HSV (Herpes simplex virus), an antigen of Neisseria meningitidis, an antigen of Corynebacterium diphtheria, an antigen of Bordetella pertussis, and Clostridium. Clostridium tetani antigen, human papilloma virus antigen, 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), severe acute respitaroty syndrome-corona virus;A vaccine composition comprising at least one selected from the group consisting of an antigen of severe acute respiratory syndrome coronavirus (SARS-CoV), an antigen of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), an antigen of influenza A virus, an antigen of influenza B virus, an antigen of Ebola virus, and an antigen of Streptococcus pneumoniae.; 10. A 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 combined vaccine.

11. A composition according to claim 1, wherein the vaccine composition comprises a fusion protein in which the antigen protein and the immune-enhancing protein are linked, or a polynucleotide encoding the same.

12. An immune-enhancing or immune-adjuvant composition comprising a protein derived from a bacterial or viral glycoprotein or a polynucleotide encoding the same.

13. A polypeptide for enhancing antibody formation ability, comprising a protein derived from a glycoprotein of bacteria and viruses.

14. A polynucleotide comprising a polynucleotide encoding a peptide for enhancing antibody formation ability according to claim 13.

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