WO2021201612A1 - Novel vaccine composition for prevention and treatment of coronavirus - Google Patents

Abstract

The present invention relates to a novel vaccine composition for preventing and treating CoV. More specifically, the present invention provides a vaccine composition for preventing and treating CoV comprising, as an active ingredient, an S1 glycoprotein of CoV or an immunogenic fragment thereof, or a polynucleotide encoding the S1 glycoprotein or an immunogenic fragment thereof.

Description

Vaccine composition for prevention and treatment of novel coronavirus

The present invention relates to a vaccine composition, and more particularly, to a vaccine composition for preventing and treating novel coronaviruses.

Due to the recent global pandemic of COVID-19, humanity is facing a serious public health crisis. Although there is no problem with COVID-19 in healthy people, it can pose a great threat to the elderly or those with underlying diseases, and sometimes even in healthy and young people, when an abnormal immune response such as a cytokine storm is induced, it is a very serious situation. can cause Coronavirus (hereinafter abbreviated as “CoV”) is a spike glycoprotein (abbreviated as “S glycoprotein” hereinafter), hemagglutinin-acetyltransferase glycoprotein, membrane glycoprotein and small envelope It is composed of several glycoproteins, such as glycoproteins (Fig. 1). The S glycoprotein mediates receptor recognition and membrane fusion of coronaviruses and serves as a primary target for humoral immune responses during infection. The CoV S glycoprotein consists of two subunits, one containing the receptor binding domain (RBD) (S1) and the other, the fusion machinery subunit (S2). So far, only S glycoprotein is known to be able to induce neutralizing antibodies, which is a decisive effector against coronavirus ( FIG. 2 ).

Recently, multinational pharmaceutical companies such as Pfizer, AstraZeneca, and Moderna have developed vaccines for preventing COVID-19, and these vaccines have passed emergency clinical trials and are being sold and vaccinated. However, in the case of AstraZeneca's vaccine, a number of side effects such as fever, chills, and muscle pain occur, and in the case of the elderly with underlying diseases, fatal accidents occur in severe cases, making it a burden for the elderly to be vaccinated, In the case of Pfizer's vaccine, the vaccine defense efficiency is higher and side effects are relatively few compared to AstraZeneca's vaccine, but the probability of side effects is 10 times higher than that of the flu vaccine, and because it is an mRNA vaccine, it is difficult to store and transport. There is a problem that there is a lot of potential for problems to occur.

There are many vaccine compositions for preventing CoV infection in other animals, such as dogs and cattle, other than humans. For example, Korean Patent No. 1242757 discloses a spike recombinant protein comprising a canine CoV neutralizing antigen determinant and a vaccine composition comprising the same, and Japanese Patent No. 4160201 discloses DNA encoding a feline infectious peritonitis virus nucleic acid capsid protein. Disclosed is a DNA vaccine for preventing corona virus infection that can confer immunity against cat or canine corona virus containing Vaccine is being launched.

On the other hand, there are some vaccines for preventing CoV infecting humans. Most of these vaccines target the severe acute respiratory syndrome (SARS) CoV that was prevalent in the mid-2000s. Whether or not it will work against SARS CoV-2, which is prevalent in 2020 It is unknown.

Therefore, there is an urgent need for the development of a more effective vaccine that enables immunization against SARS CoV-2 suitable for the human body.

The present invention aims to solve various problems including the above problems, and to provide a vaccine composition for the prevention and treatment of CoV including CoV 19. However, the scope of the present invention is not limited by the above purpose.

According to one aspect of the present invention, there is provided a vaccine composition for preventing and treating SARS Cov containing a polynucleotide encoding a water-soluble fragment polypeptide of the S glycoprotein of SARS Cov as an active ingredient.

According to one aspect of the present invention, there is provided a vaccine composition for preventing and treating SARS CoV comprising a polynucleotide encoding a fusion protein comprising a water-soluble fragment polypeptide of the S glycoprotein of SARS Cov and a trimer forming protein as an active ingredient do.

According to another aspect of the present invention, there is provided a method for preventing and treating SARS-CoV infection in the subject, comprising administering the vaccine composition to the subject.

The vaccine composition according to an embodiment of the present invention can be effectively used for the prevention and treatment of SARS-CoV infection by not only exhibiting a high antibody titer value in the inoculated individual but also effectively causing the individual's T cell response.

1 is a schematic diagram schematically showing the virion structure of CoV.

Figure 2 is a schematic diagram schematically showing the domain structure of the spike glycoprotein of CoV.

3 is a representative amino acid sequence of the COVID19 S glycoprotein derived by the present inventors.

Figure 4 is the result of confirming the signal sequence position in the representative amino acid sequence of the COVID19 S glycoprotein derived by the present inventors through the prediction program.

5 is a result showing the position of each domain in the representative amino acid sequence of the COVID19 S glycoprotein derived by the present inventors in the amino acid sequence.

6 is a genetic map schematically showing various structures of a DNA vaccine for CoV prevention and infection according to an embodiment of the present invention.

7 is a recombinant protein trimerization strategy using T4 bacteriophage fibritin (A), surfectin D protein (B) and CD40L (C), which are trimer-forming recombinant proteins used for inducing trimerization of S glycoprotein of the present invention. It is a schematic schematic diagram.

8 is a result of confirming whether the protein is expressed from the COVID19 vaccine construct according to an embodiment of the present invention.

9 to 11 show the experimental method and the experimental results confirming the efficacy (antibody response, T cell response) of the COVID19 vaccine in a mouse model.

12a is a schematic diagram showing the structure of a gene construct prepared to express various COVID19 antigens (pGX27-S: S1S2 _full , pGX27-S _ΔTM : S1S2 _{ΔTM/IC) prepared according to an embodiment of the present invention;} , Figure 12b is a photograph showing the result of analyzing the expression of SARS CoV-2 antigen produced in cells transformed with the gene construct by Western blot analysis.

13 is a schematic diagram showing administration information (top) and administration schedule for mice (bottom) of the DNA vaccine composition according to an embodiment of the present invention.

14 is a graph showing the results of measuring the immune response after administration of various vaccine compositions prepared according to an embodiment of the present invention to a mouse model.

According to one aspect of the present invention, there is provided a vaccine composition for preventing and treating SARS Cov containing a polynucleotide encoding a water-soluble fragment polypeptide of S glycoprotein of SARS Cov (Severe acute respiratory syndrome coronavirus) as an active ingredient.

As used herein, "a water-soluble fragment of SARS Cov S glycoprotein" refers to a polymorph in which the transmembrane domain (TM) and/or intracellular domain (IC) portion of the envelope glycoprotein of SARS coronavirus has been removed. Peptide fragments, fragments produced by cleavage of the polypeptide fragment by proteolytic enzymes (eg, S1 protein and S2 protein), or fragments containing the receptor binding domain of S1 protein, which are water-soluble rather than membrane-bound. It refers to the smallest fragment that induces an immune response to be expressed.

In the vaccine composition, the SARS Cov may be SARS Cov-1 or SARS Cov-2, but is preferably SARS Cov-2.

In the vaccine composition, the water-soluble fragment polypeptide comprises i) a fragment comprising the S1 protein or its receptor binding domain, or ii) a fragment comprising the S1 protein or its receptor binding domain, and an S2 protein from which the transmembrane domain has been removed. It may be a fusion protein comprising

In this case, the receptor binding domain may include a polypeptide corresponding to amino acid residues 319 to 541 based on the SARS Cov-2 S glycoprotein (SEQ ID NO: 40), and the transmembrane domain may include a SARS Cov-2 S glycoprotein (SEQ ID NO: 40). It may be a polypeptide corresponding to amino acid residues 1214 to 1234 on a protein basis. The S2 protein from which the transmembrane domain is removed may be a polypeptide corresponding to amino acid residues 685 to 1213 based on SARS Cov-2 S glycoprotein or an immunogenic fragment thereof. The water-soluble fragment polypeptide may include a polypeptide corresponding to amino acid residues 16 to 1213 based on SARS Cov-2 S glycoprotein.

The vaccine composition may include a water-soluble fragment polypeptide of SARS Cov S glycoprotein as an active ingredient instead of the polynucleotide.

According to one aspect of the present invention, there is provided a vaccine composition for preventing and treating SARS CoV comprising a polynucleotide encoding a fusion protein comprising a water-soluble fragment polypeptide of the S glycoprotein of SARS Cov and a trimer forming protein as an active ingredient do.

In the vaccine composition, the S1 glycoprotein may include the amino acid sequence set forth in SEQ ID NO: 1.

In the vaccine composition, the polynucleotide encoding the S1 glycoprotein may include any one of the nucleic acid sequences set forth in SEQ ID NOs: 27 to 29.

In the vaccine composition, the S2 glycoprotein or immunogenic fragment thereof may have a transmembrane domain and an intracellular domain removed.

In the vaccine composition, the S2 glycoprotein may include an amino acid sequence set forth in SEQ ID NO: 2.

The S2 glycoprotein may be encoded by a polynucleotide having a nucleic acid sequence set forth in SEQ ID NO: 30 or 31.

In the vaccine composition, the polynucleotide encoding the fusion protein comprising the S1 glycoprotein and the S2 glycoprotein may include a nucleic acid sequence set forth in SEQ ID NO: 36 or 37.

In the vaccine composition, the trimer forming protein may be CD40L ectodomain, T4 bacteriophage fibritin, Fas ligand, 41BB, OX40L, CD70, TRAIL, or serfectin, and the CD40L ectodomain is N - It may be a soluble CD40L (sCD40L) from which the terminal domain (1-112 aa) has been removed, and may include the amino acid sequence shown in SEQ ID NO: 3. The surfectin may be surfectin A or surfectin D.

In the vaccine composition, the polynucleotide encoding the fusion protein comprising the S1 glycoprotein and the CD40L protein may include the nucleic acid sequence set forth in SEQ ID NO: 38.

In the vaccine composition, the S1 glycoprotein, and the S2 glycoprotein; And the polynucleotide encoding the fusion protein comprising the trimer forming protein may include a nucleic acid sequence set forth in SEQ ID NO: 38 or 39.

As used herein, the term “immunogenic fragment” refers to a fragment capable of inducing an immune response in vivo among fragments generated by cleaving an antigenic protein. The smallest unit of such a fragment may be an epitope.

As used herein, the term “fusion protein” refers to a recombinant protein in which two or more proteins or domains responsible for specific functions within the protein are linked. A linker peptide having a generally flexible structure may be inserted between the two or more proteins or domains, if it is a flexible peptide linker that does not limit the original function of the linked polypeptide and does not inhibit the expression of the fusion protein Any one can be used, and specific examples are as described above.

In the composition, the S2 glycoprotein or immunogenic fragment thereof may have a transmembrane domain and an intracytoplasmic domain removed. The CD40L ectodomain may be soluble CD40L (sCD40L) from which the N-terminal domain (1-112 a.a) has been removed.

When the vaccine composition includes a polynucleotide encoding a fusion protein, the fusion protein may include one or more linker peptides between fusion partners, and any known linker peptide may be used. Examples of such linkers include (GS) ₅ (SEQ ID NO: 4), (G ₄ S, SEQ ID NO: 5) _n , (GSSGGS, SEQ ID NO: 6) _n , KESGSVSSEQLAQFRSLD (SEQ ID NO: 7), EGKSSGSGSESKST (SEQ ID NO: 8), GSAGSAAGSGEF (SEQ ID NO: 9), (EAAAK, SEQ ID NO: 10) n, CRRRRRREAEAC (SEQ ID NO: 11), A (EAAAK) ₄ ALEA (EAAAK) ₄ A (SEQ ID NO: 12), GGGGGGGG (SEQ ID NO: 13), GGGGGG (SEQ ID NO: 11) 14), AEAAAKEAAAAKA (SEQ ID NO: 15), PAPAP (SEQ ID NO: 16), (Ala-Pro) _n , VSQTSKLTRAETVFPDV (SEQ ID NO: 17), PLGLWA (SEQ ID NO: 18), TRHRQPRGWE (SEQ ID NO: 19), AGNRVRRSVG (SEQ ID NO: 20) ), RRRRRRRR (SEQ ID NO: 21), GFLG (SEQ ID NO: 22), and GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE (SEQ ID NO: 23) can be used.

In addition, the S1 protein or the fusion protein may include a secretion signal sequence for protein secretion at the N-terminus. Any known secretion signal sequence may be used, for example, a tissue plasminogen activator (tPA) signal sequence (SEQ ID NO: 24), an HSV gDs (herpes simplex virus glycoprotein Ds) signal sequence, or a growth hormone signal sequence. .

As used herein, the term "operably linked to" means that a nucleic acid sequence of interest (eg, in an in vitro transcription/translation system or in a host cell) is regulated in such a way that its expression can be achieved. It means that it is connected to the sequence. The term "regulatory sequence" is meant to include promoters, enhancers and other regulatory elements (eg, polyadenylation signals). Regulatory sequences include instructing that a target nucleic acid can be constitutively expressed in many host cells, instructing the expression of a target nucleic acid only in specific tissue cells (eg, tissue-specific regulatory sequences), and This includes directing expression to be induced by a specific signal (eg, an inducible regulatory sequence). It can be understood by those skilled in the art that the design of the expression vector may vary depending on factors such as the selection of the host cell to be transformed and the level of desired protein expression. The expression vector of the present invention can be introduced into a host cell to express the fusion protein. Regulatory sequences enabling expression in eukaryotic and prokaryotic cells are well known to those skilled in the art. As described above, they usually contain regulatory sequences responsible for initiation of transcription and, optionally, poly-A signals responsible for termination and stabilization of transcripts. Additional regulatory sequences may include, in addition to transcriptional regulators, translation enhancers and/or natively-combined or heterologous promoter regions. Possible regulatory sequences enabling expression in mammalian host cells, for example, include the CMV-HSV thymidine kinase promoter, SV40, RSV (Rous sarcoma virus)-promoter, human elongation element 1α (hEF1α)-promoter, glucocorticoid-inducing promoters such as the sexual MMTV-promoter (Moloni mouse tumor virus), metallothionein-inducible or tetracycline-inducible promoter, or CMV promoter or SV40 promoter. For expression in neurons, it is contemplated that neurofilament-promoter, PGDF-promoter, NSE-promoter, PrP-promoter or thy-1-promoter may be used. Such promoters are known in the art and are described in Charron, J. Biol. Chem. 270: 25739-25745, 1995. For expression in prokaryotes, a number of promoters have been disclosed, including the lac promoter, the tac promoter or the trp promoter. In addition to factors capable of initiating transcription, the regulatory sequences include transcription termination signals such as SV40-poly-A site or TK-poly-A site downstream of the polynucleotide according to an embodiment of the present invention. You may. In this document, suitable expression vectors are known in the art, for example, Okayama-Berg cDNA expression vectors pcDV1 (Parmacia), pRc/CMV, pcDNA1, pcDNA3 (In-vitrogene), pSPORT1 (GIBCO BRL) ), pGX27 (Patent No. 1442254), pX (Pagano, Science 255: 1144-1147, 1992), yeast two-hybrid vectors such as pEG202 and dpJG4-5 (Gyuris, Cell 75: 791-803) , 1995) or prokaryotic expression vectors such as lambda gt11 or pGEX (Amersham-Pharmacia). In addition to the nucleic acid molecules of the present invention, the vector may further comprise a polynucleotide encoding a secretion signal. The secretion signals are well known to those skilled in the art. And, depending on the expression system used, a leader sequence capable of directing the fusion protein to the cell compartment is combined with the coding sequence of the polynucleotide according to an embodiment of the present invention, preferably the translated protein or its It is a leader sequence capable of directly secreting a protein into the periplasmic or extracellular medium.

The vaccine composition may further comprise a polynucleotide encoding one or more immuno-enhancing peptides, in which case the immuno-enhancing peptide may be expressed in the form of a fusion protein linked to the S1 glycoprotein or fusion protein, In this case, the immuno-enhancing peptide may be added to the N-terminus or C-terminus of the S1 glycoprotein or the fusion protein or inserted in the middle, and the immuno-enhancing peptide may be CD28, ICOS (inducible costimulator), CTLA4 (cytotoxic T lymphocyte). associated protein 4), PD1 (programmed cell death protein 1), BTLA (B and T lymphocyte associated protein), DR3 (death receptor 3), 4-1BB, CD2, CD40, CD40L, CD30, CD27, SLAM (signaling lymphocyte activation) molecule), 2B4 (CD244), NKG2D (natural-killer group 2, member D)/DAP12 (DNAX-activating protein 12), TIM1 (T-Cell immunoglobulin and mucin domain containing protein 1), TIM2, TIM3, TIGIT, CD226 , CD160, LAG3 (lymphocyte activation gene 3), B7-1, B7-H1, GITR (glucocorticoid-induced TNFR family related protein), Flt3 ligand (fms-like tyrosine kinase 3 ligand), flagellin, HVEM ( herpesvirus entry mediator) or the cytoplasmic domain of OX40L [ligand for CD134 (OX40), CD252], or a linkage of two or more thereof.

In the composition, the Flt3 ligand may be a polypeptide consisting of SEQ ID NO: 25.

In addition, the gene construct may be provided in the form of an expression vector such as a plasmid vector for convenience of manufacture and manipulation.

In addition, the vector used in the present invention can be prepared by, for example, standard recombinant DNA technology, and standard recombinant DNA technology includes, for example, blunt-end and adherent-end ligation, restriction enzyme treatment to provide an appropriate end. , phosphate group removal by alkaline phosphatase treatment and enzymatic ligation by T4 DNA ligase to prevent improper binding. The vector of the present invention can be prepared by recombination of a DNA encoding a signal peptide obtained by chemical synthesis or genetic recombination technology, or a DNA encoding a fusion protein according to an embodiment of the present invention, into a vector containing an appropriate regulatory sequence. have. The vector containing the control sequence can be purchased or manufactured commercially, and in an embodiment of the present invention, pGX27 (Korean Patent No. 1442254), which is a vector for preparing a DNA vaccine, was used.

The expression vector according to an embodiment of the present invention may be an expression vector capable of expressing the fusion protein in a host cell, and the expression vector is a plasmid vector, a viral vector, a cosmid vector, a phagemid vector, or an artificial human. Any form, such as chromosomes, may be represented.

According to another aspect of the present invention, there is provided a transformed host cell in which the host cell is transformed with the vector. The host cell may be a bacterium, a fungus, a plant cell, or an animal cell, the bacterium may be a gram-negative bacteria or a gram-positive bacteria, and the animal cell may be an insect cell or a mammalian cell. The mammalian cells may be primary cells or established cell lines isolated from rats, mice, monkeys, hamsters, dogs, horses, cattle, cats, pigs, elephants, monkeys, primates or humans. Such host cells can be used for the production of antigenic proteins that can be used in vaccines by transformation of the gene construct according to an embodiment of the present invention.

The vaccine composition may include one or more pharmaceutically acceptable vaccine adjuvants. The vaccine adjuvant includes aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), MF59, virosome, AS04 [a mixture of aluminum hydroxide and monophosphoryl lipid A (MPL)], AS03 ( DL-α-tocopherol, a mixture of squalene and the emulsifier polysorbate 80), CpG, flagellin, Poly I:C, AS01, AS02, ISCOMs and ISCOMMATRIX, etc. can be used. As used herein, the term “adjuvant” or “vaccine adjuvant” refers to a pharmaceutical or immunological agent administered for the purpose of enhancing the immune response of a vaccine.

Optionally, the vaccine composition comprises IL-12 protein and IL-21 protein as active ingredients together with or instead of the above-described vaccine adjuvant, or a polynucleotide encoding the IL-12 protein and a polynucleotide encoding the IL-21 protein. It may include a vaccine adjuvant for promoting a T lymphocyte-specific immune response comprising a polynucleotide as an active ingredient.

The composition may further include a pharmaceutically acceptable adjuvant, excipient or diluent in addition to the carrier.

As used herein, the term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not normally cause gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions when administered to humans. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, fillers, anti-agglomeration agents, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.

In addition, the vaccine composition according to an embodiment of the present invention may be formulated using methods known in the art to enable rapid, sustained or delayed release of the active ingredient when administered to a mammal. Formulations include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, and sterile powder forms.

The vaccine composition according to an embodiment of the present invention may be administered by various routes, for example, oral, parenteral, for example, suppository, transdermal, intravenous, intraperitoneal, intramuscular, intralesional, nasal, intravertebral administration In addition, it can be administered using an implantation device for sustained release or continuous or repeated release. The number of administration may be administered once a day or divided into several times within a desired range, and the administration period is not particularly limited.

The vaccine composition according to an embodiment of the present invention may be administered by general systemic administration or local administration, for example, intramuscular injection or intravenous injection, but when provided as a DNA vaccine composition, most preferably an electroporator can be injected using The electroporation machine is a commercially available electroporator for DNA drug injection, such as Glinporator ^TM of IGEA of Italy, CUY21EDIT of JCBIO of Korea, SP-4a of Supertech of Switzerland, OrbiJector ^® of SL Vaxigen (Korea), etc. can be used

The route of administration of the vaccine composition according to an embodiment of the present invention may be administered through any general route as long as it can reach the target tissue. Such administration route may be parenteral administration, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intrasynovial administration, but is not limited thereto.

The vaccine composition according to an embodiment of the present invention may be formulated in a suitable form together with a commonly used pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, carriers for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycol, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. In addition, the composition according to the present invention may contain a suspending agent, a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjuster, an analgesic agent, a buffer, Antioxidants and the like may be included as appropriate. Pharmaceutically acceptable carriers and agents suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, latest edition.

In addition, the vaccine composition of the present invention is administered in an amount necessary for prevention or a therapeutically effective amount.

As used herein, the term "therapeutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and effective dose level includes the subject type and severity, age, sex, drug activity, sensitivity to drugs, administration time, administration route and excretion rate, duration of treatment, factors including concurrent drugs, and other factors well known in the medical field. The vaccine composition or pharmaceutical composition of the present invention may be administered at a dose of 0.05 mg/dose to 100 mg/dose, more preferably 0.1 mg/dose to 10 mg/dose. Meanwhile, the dosage may be appropriately adjusted according to the patient's age, sex, and condition.

According to another aspect of the present invention, there is provided a method for preventing and treating SARS-CoV infection in the subject, comprising administering the vaccine composition to the subject.

In the treatment method, the SARS CoV may be Severe Acute Respiratory Syndrome (SARS) Cov-1, or SARS Cov-2, in particular, SARS Cov-2 is a novel coronavirus disease (COVID-19) that is currently prevalent worldwide. 30 January 2020 the World Health Organization declared Coronavirus Disease 2019).

In the above method, the vaccine composition may be administered by in vivo electroporation.

Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. However, the present invention is not limited to the examples and experimental examples disclosed below, but can be implemented in various different forms, and the following examples and experimental examples make the disclosure of the present invention complete, and to which the present invention belongs. It is provided to fully inform those of ordinary skill in the art the scope of the invention.

Example: Design of CoV DNA Vaccines

In order to prepare a vaccine composition that can induce an effective immune response using CoV S glycoprotein as an antigen, it is necessary to design an antigen gene construct of CoV S glycoprotein, but SARS Cov-2 S, the pathogenic virus of COVID-19 Specific positional information for the amino acid sequence and individual domains of glycoproteins is not yet known.

Therefore, in designing the antigen gene construct, the present inventors consider that the most important point in developing an effective vaccine is to determine to what position the antigen is designed based on the sequence and the position of the domain. Based on 45 SARS-CoV-2 nucleotide sequences identified in the National Institutes of Health (NIH) gene sequence database, GenBank (http://www.ncbi.nlm.nih.gov/genbank/sars-cov-2-seqs). A representative amino acid sequence of the S glycoprotein of CoV was derived using Jalview software (http://www.jalview.org/) (FIG. 3). The representative amino acid sequence of the S glycoprotein of CoV identified by the present inventors was confirmed to be 100% identical to the NCBI reference sequence (NCBI Reference Sequence: YP_009724390.1) disclosed later. The amino acid positions of the domains of the CoV S glycoprotein were determined based on SEQ ID NO: 40, which is the same as the representative sequence.

Then, another CoV genus (CoV genus; Middle East respiratory syndrome-related coronavirus (isolated United Kingdom/H123990006) in which the S glycoprotein domain structure for the position of the S1 subunit, S2 subunit, and transmembrane domain (TM) has already been identified /2012) (Betacoronavirus England 1) (Human coronavirus EMC), Human SARS coronavirus (SARS-CoV, Severe acute respiratory syndrome coronavirus), Human coronavirus OC43 (HCoV-OC43), Human coronavirus HKU1 (isolate N5) (HCoV-HKU1) , Human coronavirus 229E (HCoV-229E), Human coronavirus NL63 (HCoV-NL63)) and at the same time comparing and analyzing the domain sequences of the literature (Kleine-Weber H et al ., Sci. Rep . 8(1): 16597, 2018 ) to determine the position of each domain based on the representative amino acid sequence of the derived S glycoprotein by synthesizing both the conserved amino acid sequence information revealed through and SignalP-5.0 software-based signal sequence cleavage site prediction information (FIG. 4) was done (FIG. 5).

Then, based on the determined domain information described in FIG. 5, four types of gene constructs were devised (Table 1 and FIG. 6). Specifically, the design was divided into a form including only the S1 subunit (FIG. 6a, Example 1) or a form including both S1/S2 subunits (FIG. 6b, Example 2). In the case of the S2 subunit, the transmembrane domain and the intracellular tail (IC) part were designed to be removed so that the antigen expressed in the cell can be effectively exposed outside the cell (S2 _ΔTM/IC ).

In addition, in order to induce an effective neutralizing antibody response, it is very important to form and expose the antigen in an intact form in the body. Since the CoV S glycoprotein exists in a trimer pre-fusion form, the CoV S glycoprotein produced in the vaccine must be in the trimer pre-fusion form. There may be several methods for trimerizing the target protein, for example, a method of linking a T4 bacteriophage fibritin trimerization motif (FT) or surfactant protein D (SPD). There is (Fig. 7).

The present inventors have devised a method in which the S glycoprotein of the CoV DNA vaccine can not only be produced in a trimeric pre-fusion form, but also have a structure capable of increasing the immune response at the same time. Specifically, CD40 ligand (CD40L), one of the ligands of the tumor necrosis factor receptor superfamily (TNFRSF), is known to signal transduction to CD40 in a trimeric form, and has been shown to increase not only the antibody immune response but also the cellular immune response. is known Therefore, the present inventors designed a vaccine comprising a polynucleotide encoding a fusion protein in the form of linking CoV S glycoprotein and soluble CD40L, so that the CoV S glycoprotein is efficiently expressed in the trimeric pre-fusion form, as well as the vaccine to increase the immune response of (Figs. 6c and 6d, Examples 3 and 4).

Example	secretory signal sequence (corresponding nucleic acid sequence)	antigen (corresponding nucleic acid sequence)	linker peptide (corresponding nucleic acid sequence)	trimerization domain (corresponding nucleic acid sequence)
One	tPA (26)	S1(27)	-	-
2	tPA (26)	S1/S2 △TM/IC (36)	-	-
3	tPA (26)	S1(29)	(GS) 5 (32)	sCD40L 113-261 (34)
4	tPA (26)	S1/S2 △TM/IC (37)	(GS) 5 (33)	sCD40L 113-261 (35)

After synthesizing the CoV DNA vaccine gene construct designed as in Examples 1 to 4, an expression vector for a DNA vaccine was prepared by inserting it into the pGX27 vector (Genexine, Inc., Korea) (FIG. 6), which It was called a DNA vaccine.

Experimental Example 1: Confirmation of protein expression by COVID-19 DNA vaccine

COS-7 cells 1 x 10 ⁷ cells were seeded in a 100 mm culture dish and cultured for 16 hours. After 24 μg of 4 types of COVID-19 DNA vaccine and 60 μL of lipofectamine 2000 were mixed and added to the cells, 37 ℃ CO _{2 In an} incubator for 4 to 5 hours, the gene was introduced and the culture medium was exchanged. After culture for 48 hours after medium exchange, the cultured cells were lysed to extract proteins. Then, 4x SDS sample buffer was mixed with the extracted protein and boiled at 72 °C for 10 minutes. After that, electrophoresis was performed using SDS gel, and the protein on the gel was moved using iBlot Gel transfer stacks nitrocellulose (Invitrogen), and then an antibody specific for SARS-CoV-2 spike was used as the primary antibody at 4°C. overnight, and the HRP-conjugated secondary antibody was diluted 1:5000 and reacted at room temperature for 1 hour. The protein product expressed by the gene introduced into the COVID-19 DNA vaccine was detected by developing the band reactive with the above antibody with ECL (Enhanced chemiluminence solution) solution. As a result, the SARS-CoV-2 full-length S protein or the S1 subunit protein in the COS-7 sample introduced with the COVID-19 DNA vaccine was confirmed through a specific band at the predicted position when expressed using the CMV promoter, and negative No specific protein was detected in the control group (FIG. 8). Through this, it was confirmed that the expression system of the antigen gene construct of the present invention operates normally.

Experimental Example 2: Evaluation of the efficacy of the COVID-19 vaccine of the present invention in a mouse model

In order to confirm the immune response in the mouse model of the COVID-19 DNA vaccine of the present invention, a mouse experiment was performed based on the administration group and schedule described in FIG. 9 .

2-1: Antibody reaction

50 μL/head of the administration was administered to the femoral muscle of BALB/c mice, and the administration was repeated twice at an interval of 2 weeks. In all administration groups, EP (200 V/cm, (3 pulses at 10 ms/pulse and 10 ms between pulses) x 4 cycles) was performed using an EP device with an EP cartridge injection needle inserted. The immunogenicity of the COVID-19 vaccine was evaluated by performing antigen-specific IgG antibody titer ELISA in serum at 2 and 4 weeks from the time of the first administration of the vaccine.

Experimental results are based on (1) whether the vaccine immune response is increased by binding of CD40L (CD40 ligand), (2) the range of use of the S antigen component (S1 subunit or S1/S2 subunit), and (3) the frequency of administration. The analysis was divided into four comparison items, such as whether or not immunogenicity was increased. As a result of analyzing whether the vaccine immune response was increased by the binding of CD40L, there was no difference in overall antibody response due to the binding of CD40L to the S antigen, or rather, there was a tendency to decrease slightly (S1 (25 μg) vs. S1-sCD40Lm ( 25 μg), p=0.075 (2 weeks), p=0.69 (4 weeks);S1/S2 (25 μg) vs. S1/S2-sCD40Lm (25 μg), p=0.042 (2 weeks), p=0.548 (4 weeks)).

As a result of analyzing the antigen (S1+S2) specific antibody response according to the range of use of the S antigen subunit, there was no significant difference in the antibody response to the S antigen between the S1 subunit administration group and the S1/S2 subunit administration group (S1). (25 μg) vs. S1/S2 (25 μg), p=0.089 (2 weeks), p=0.841 (4 weeks); S1-sCD40Lm (25 μg) vs. S1/S2-sCD40Lm (25 μg), p =0.28(2 weeks), p=0.421(4 weeks)).

As a result of observing whether immunogenicity increased according to the number of administrations (one or two), a significant antibody titer was induced only with a single administration. There was a tendency to slightly increase during boosting, but there was no statistically significant difference (Prime-boost: S1 (25 μg), p=0.310; S1-sCD40Lm (25 μg), p=0.222; S1/S2 (25 μg). ), p=0.056;S1/S2-sCD40Lm (25 μg), p=0.032). However, the phenomenon that the antibody reaction increased after boosting of the individual with low antibody titer during priming was the same. This is a result demonstrating that the COVID-19 vaccine of the present invention is an effective vaccine that can exhibit preventive and therapeutic efficacy against viral infection by increasing the antibody response (FIG. 10).

2-2: T cell response

50 μL/head of the administration was administered to the femoral muscle of BALB/c mice, and the administration was repeated twice at an interval of 2 weeks. All administration groups were subjected to electroporation (200 V/cm, (3 pulses at 10 ms/pulse and 10 ms between pulses) x 4 cycles) using an electroporation device with an EP cartridge injection needle inserted. Two weeks after the last administration (week 4), the spleen was removed and the spike antigen-specific T cell response was analyzed using the ELISPOT method.

The efficacy of the COVID-19 vaccine was confirmed by evaluating the SARS Cov-2 spike antigen-specific T cell response by the IFN-γ ELISPOT method, and the experimental results were (1) efficacy of increasing vaccine immune response by binding of CD40L (CD40 ligand) Analysis was divided into evaluation, (2) determining the range of use of the spike protein subunit (S1 subunit or S1/S2 subunit).

As a result of analyzing the vaccine-specific T cell immune response induction ability with or without CD40L, there was no difference according to the binding of CD40L to the S antigen (S1 vs. S1-sCD40Lm, p=0.151; S1/S2 vs. S1S2-sCD40Lm, p = 0.841), as a result of analyzing the vaccine-specific T-cell immune response according to the range of use of the S antigen subunit, the S1/S2 subunit-administered group showed a positive effect on the T cell response to the entire spike protein pool compared to the S1 subunit. It showed an increasing trend (S1 vs. S1/S2, p=0.548; S1-sCD40Lm vs. S1/S2-sCD40Lm, p=0.056) (FIG. 11).

The coronavirus to which SARS Cov-2 belongs infects the host cell through angiotensin-converting enzyme 2 (ACE2), and it is known that the receptor binding domain (RBD) is located in the S1 subunit. Therefore, it is understood that it is important to induce an antibody response well to the S1 subunit. Four types of COVID-19 DNA vaccines designed by the present inventors (pGX27-COVID-19/S1, pGX27-COVID-19/S1S2, pGX27-COVID-19/S1-CD40L and pGX27-COVID-19/S1S2-CD40L) The antibody response was at a similar level, indicating that the antibody response to the vaccine was mainly directed to the S1 subunit. At this time, it is also very important to help the rapid recovery after infection through the efficient removal of virus-infected cells through the T cell reaction as well as the antibody reaction. It was confirmed that all of them were well induced, and through this, it was confirmed that a large number of T cell epitopes related to the therapeutic immune response could be distributed in the S2 subunit.

Therefore, it is thought that a construct capable of inducing a similar antibody response and at the same time inducing efficient T cells for the S1 and S2 subunits would be more appropriate for application as a vaccine. It was confirmed that pGX27-COVID-19/S1S2 without structural alteration linking CD40L to the antigen containing all units is the optimal DNA vaccine construct form for COVID-19.

Experimental Example 3: Comparison of immunogenicity according to the form of COVID-19 S glycoprotein antigen

Through the above Experimental Examples 2 and 3, it was confirmed that pGX27-COVID-19/S1S2 as a COVID-19 vaccine is an optimal vaccine construct for the prevention and treatment of COVID-19 infection. At this time, the S2 subunit includes a hepted repeat region involved in trimerization and a transmembrane domain penetrating the surface membrane. In order to confirm the effect on immunogenicity, a gene construct including the S2 subunit in the full-length form of the S1 protein antigen was additionally designed as shown in FIG. 12a.

To determine whether the gene construct is normally expressed, the gene constructs were transfected into COS-7, and protein expression patterns were analyzed by Western blot analysis using an anti-S glycoprotein antibody. As a result, as shown in FIG. 12B , it was confirmed that both the S1S2 _full gene construct and the S1S2 _ΔTM/IC gene construct normally express the S glycoprotein. However, the expression level of the S glycoprotein in the form in which the transmembrane domain has been removed was significantly increased. This is interpreted to be because the S glycoprotein is produced in the form of a water-soluble protein rather than a membrane-bound form.

Then, in order to confirm the immune response in the mouse model of the COVID-19 DNA vaccine according to the shape of the S1 subunit, an experiment was performed based on the administration group and schedule described in FIG. 13 .

Specifically, 50 μL/head of the administration was administered to the femoral muscle of BALB/c mice, and the administration was repeated twice at an interval of 2 weeks. All administration groups were subjected to electroporation (200 V/cm, (3 pulses at 10 ms/pulse and 10 ms between pulses) x 4 cycles) using an electroporation device with an EP cartridge injection needle inserted. Antigen-specific IgG antibody titer ELISA in serum was performed at the 2nd and 4th weeks from the time of the first vaccine administration to evaluate the immunogenicity of the COVID-19 vaccine candidate.

As a result, the antibody response after one administration did not differ significantly depending on the antigen type, but the S1S2 _ΔTM/IC type vaccine showed the highest antibody titer value, and after the second administration, the antibody response of the S1S2 _ΔTM/IC type vaccine showed the highest antibody titer value. It was confirmed that the vaccine administration group showed a statistically significantly higher antibody titer value than the other two types of vaccine administration group (FIG. 14).

In conclusion, it was found that the form in which the transmembrane domain of the S2 subunit was removed while simultaneously applying the S1 subunit and the S2 subunit as a COVID19 vaccine antigen was a more effective vaccine construct, thereby completing the present invention.

Although the present invention has been described with reference to the above-described examples and experimental examples, it will be understood that these are merely exemplary, and that various modifications and equivalent other embodiments are possible therefrom by those skilled in the art. Therefore, the true technical protection scope of the present invention should be determined by the technical spirit of the appended claims.

The vaccine composition according to an embodiment of the present invention can be used to develop a medicament for preventing and treating SARS CoV infection.

Claims (35)

1. A vaccine composition for the prevention and treatment of SARS Cov comprising a polynucleotide encoding a water-soluble fragment polypeptide of the S glycoprotein of SARS Cov as an active ingredient.
2. According to claim 1,

   The SARS Cov is SARS Cov-1 or SARS Cov-2, the vaccine composition.
3. According to claim 1,

   The water-soluble fragment polypeptide is

   i) a fragment comprising an S1 protein or a receptor binding domain thereof; or

   ii) a fusion protein comprising the S1 protein or a fragment containing the receptor binding domain thereof and the S2 protein from which the transmembrane domain has been removed, the vaccine composition.
4. According to claim 1,

   Wherein the receptor binding domain comprises a polypeptide corresponding to amino acid residues 319 to 541 th based on the SARS Cov-2 S glycoprotein.
5. 4. The method of claim 3,

   The transmembrane domain is a polypeptide corresponding to amino acid residues 1214 to 1234 based on the SARS Cov-2 S glycoprotein, vaccine composition.
6. 4. The method of claim 3,

   The S2 protein from which the transmembrane domain is removed is a polypeptide or an immunogenic fragment thereof corresponding to amino acid residues 685 to 1213 based on SARS Cov-2 S glycoprotein, or an immunogenic fragment thereof.
7. 4. The method of claim 3,

   The water-soluble fragment polypeptide comprises a polypeptide corresponding to amino acid residues 16-1213 based on SARS Cov-2 S glycoprotein, vaccine composition.
8. 8. The method according to any one of claims 1 to 7,

   The S1 protein is a vaccine composition comprising the amino acid sequence set forth in SEQ ID NO: 1.
9. According to claim 1,

   The polynucleotide is a vaccine composition comprising any one of the nucleic acid sequences set forth in SEQ ID NOs: 27 to 29.
10. 4. The method of claim 3,

    S2 protein from which the transmembrane domain is removed comprises the amino acid sequence set forth in SEQ ID NO: 2, vaccine composition.
11. A vaccine composition for preventing and treating SARS-CoV comprising, as an active ingredient, a polynucleotide encoding a fusion protein comprising a water-soluble fragment polypeptide of the S glycoprotein of SARS Cov and a trimer-forming protein.
12. 12. The method of claim 11,

    The SARS Cov is SARS Cov-1 or SARS Cov-2, vaccine composition
13. 12. The method of claim 11,

    The water-soluble fragment polypeptide is

    i) a fragment comprising an S1 protein or a receptor binding domain thereof; or

    ii) a fusion protein comprising the S1 protein or a fragment containing the receptor binding domain thereof and the S2 protein from which the transmembrane domain has been removed, the vaccine composition.
14. 12. The method of claim 11,

    Wherein the receptor binding domain comprises a polypeptide corresponding to amino acid residues 319 to 541 th based on the SARS Cov-2 S glycoprotein.
15. 14. The method of claim 13,

    The transmembrane domain is a polypeptide corresponding to amino acid residues 1214 to 1234 based on the SARS Cov-2 S glycoprotein, vaccine composition.
16. 14. The method of claim 13,

    The S2 protein from which the transmembrane domain is removed is a polypeptide or an immunogenic fragment thereof corresponding to amino acid residues 685 to 1213 based on SARS Cov-2 S glycoprotein, or an immunogenic fragment thereof.
17. 14. The method of claim 13,

    The water-soluble fragment polypeptide comprises a polypeptide corresponding to amino acid residues 16-1213 based on SARS Cov-2 S glycoprotein, vaccine composition.
18. 12. The method of claim 11,

    wherein the trimer forming protein is CD40L ectodomain, T4 bacteriophage fibritin, Fas ligand, 41BB, OX40L, CD70, TRAIL, or serfectin.
19. 19. The method of claim 18,

    The CD40L ectodomain comprises the amino acid sequence set forth in SEQ ID NO: 3, vaccine composition.
20. 19. The method of claim 18,

    The surfectin is surfectin A or surfectin D, vaccine composition.
21. 12. The method of claim 11,

    The polynucleotide comprises a nucleic acid sequence set forth in SEQ ID NO: 38 or 39, vaccine composition.
22. 14. The method of any one of claims 3 or 13,

    wherein the fusion protein comprises one or more linker peptides.
23. 23. The method of claim 22,

    The linker peptide is (GS) ₅ (SEQ ID NO: 4), (G ₄ S, SEQ ID NO: 5) _n , (GSSGGS, SEQ ID NO: 6) _n , KESGSVSSEQLAQFRSLD (SEQ ID NO: 7), EGKSSGSGSESKST (SEQ ID NO: 8), GSAGSAAGSGEF ( SEQ ID NO: 9), (EAAAK, SEQ ID NO: 10) n, CRRRRRREAEAC (SEQ ID NO: 11), A (EAAAK) ₄ ALEA (EAAAK) ₄ A (SEQ ID NO: 12), GGGGGGGG (SEQ ID NO: 13), GGGGGG (SEQ ID NO: 14 ), AEAAAKEAAAAKA (SEQ ID NO: 15), PAPAP (SEQ ID NO: 16), (Ala-Pro) _n , VSQTSKLTRAETVFPDV (SEQ ID NO: 17), PLGLWA (SEQ ID NO: 18), TRHRQPRGWE (SEQ ID NO: 19), AGNRVRRSVG (SEQ ID NO: 20) , RRRRRRRR (SEQ ID NO: 21), GFLG (SEQ ID NO: 22), or GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE (SEQ ID NO: 23).
24. 12. The method of claim 1 or 11,

    Wherein the polypeptide comprises a secretion signal sequence for protein secretion at the N-terminus, vaccine composition.
25. 25. The method of claim 24,

    The secretion signal sequence is a tPA (tissue plasminogen activator) signal sequence (SEQ ID NO: 24), HSV gDs (herpes simplex virus glycoprotein Ds) signal sequence or a growth hormone signal sequence, vaccine composition.
26. 12. The method of claim 1 or 11,

    The polynucleotide is provided as an expression vector comprising a gene construct operably linked to a promoter, vaccine composition.
27. 27. The method of claim 26,

    Said promoters are CMV-HSV thymidine kinase promoter, SV40, RSV-promoter (Loews sarcoma virus), human kidney element 1α-promoter, glucocorticoid-inducible MMTV-promoter (Moloney mouse tumor virus), metallothionein -inducible or tetracycline-inducible promoter or, CMV promoter or SV40 promoter, neurofilament-promoter, PGDF-promoter, NSE-promoter, PrP-promoter or thy-1-promoter, vaccine composition .
28. 28. The method of claim 27,

    The expression vector is Okayama-Berg cDNA expression vector pcDV1 (Parmacia), pRc/CMV, pcDNA1, pcDNA3, pSPORT1, pGX27, pX, pEG202, pJG4-5 lambda gt11, pGEX (Amersham-Pharmacia), vaccine composition.
29. 12. The method of claim 1 or 11,

    A vaccine composition, further comprising a polynucleotide encoding one or more immuno-enhancing peptides.
30. 30. The method of claim 29,

    The immune enhancing peptide is expressed in the form of a fusion protein linked to the polypeptide or is expressed through a separate gene construct, vaccine composition.
31. 31. The method of claim 30,

    The immuno-enhancing peptide is added to or inserted in the middle of the N-terminus or C-terminus of the polypeptide.
32. 30. The method of claim 29,

    The immunostimulating peptide is CD28, ICOS (inducible costimulator), CTLA4 (cytotoxic T lymphocyte associated protein 4), PD1 (programmed cell death protein 1), BTLA (B and T lymphocyte associated protein), DR3 (death receptor 3), 4 -1BB, CD2, CD40, CD40L, CD30, CD27, SLAM (signaling lymphocyte activation molecule), 2B4 (CD244), NKG2D (natural-killer group 2, member D)/DAP12 (DNAX-activating protein 12), TIM1 (T) -Cell immunoglobulin and mucin domain containing protein 1), TIM2, TIM3, TIGIT, CD226, CD160, LAG3 (lymphocyte activation gene 3), B7-1, B7-H1, GITR (glucocorticoid-induced TNFR family related protein), Flt3 ligand (fms-like tyrosine kinase 3 ligand), flagellin, a herpesvirus entry mediator (HVEM), or a cytoplasmic domain of OX40L [ligand for CD134(OX40), CD252] or a linkage of two or more thereof, a vaccine composition.
33. A method for preventing and treating SARS-CoV infection in an individual comprising administering the vaccine composition of claim 1 or 11 to the individual.
34. 34. The method of claim 33,

    The SARS CoV is SARS Cov-1 or SARS Cov-2, the method of treatment.
35. 34. The method of claim 33,

    wherein the composition is administered by in vivo electroporation.

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