Novel coronavirus recombinant spike protein, polynucleotides encoding same, vectors containing the polynucleotides, and vaccines for preventing or treating coronavirus infections comprising the vectors

Abstract

The present invention relates to a novel coronavirus recombinant spike protein, a polynucleotide encoding the same, a vector containing the polynucleotide, and a vaccine containing the vector for preventing or treating coronavirus infection. The coronavirus recombinant spike protein of the present invention is stable and therefore not easily degraded in cells, and efficiently activates immune cells, resulting in high antibody production and T cell reactivity. The vector of the present invention has been confirmed to exhibit high antigen expression levels, thereby resulting in high antibody production and T cell reactivity, a long antibody production and expression period, and no liver toxicity. Therefore, the vector of the present invention can be used to serve as a vaccine for preventing or treating coronavirus infection.

Description

[Technical field]

[0001] 1.Technical Field The present invention relates to a novel coronavirus recombinant spike protein, a polynucleotide encoding same, a vector comprising said polynucleotide, and a vaccine for preventing or treating coronavirus infection comprising said vector. [Background technology]

[0002] 2. Description of Related Art Coronaviruses, which cause severe respiratory illness and death, are classified as RNA viruses belonging to the Coronaviridae family, and coronavirus infection is defined as a respiratory syndrome caused by infection with a coronavirus. Among the viruses belonging to the Coronaviridae family, there are a total of seven types of viruses known to infect humans, including four types that cause the common cold (229E, OC43, NL63, and HKU1), two types that cause severe pneumonia (SARS-Cov and MERS-CoV), and SARS-CoV-2, the virus responsible for the current pandemic. Three types of viruses that cause severe pneumonia (SARS-CoV, MERS-CoV, and SARS-CoV-2) have spread worldwide, starting with SARS-CoV in 2002, then MERS-CoV in 2012, and now SARS-CoV-2, which has high genetic sequence homology with SARS-CoV.

[0003] Coronaviruses (family Coronaviridae) have unstable RNA as their genome, and therefore mutations occur easily, and these characteristics make them susceptible to transmission in both animals and humans. Previous studies have shown that SARS-CoV was transmitted from civet cats to humans, and MERS-CoV was transmitted from dromedary camels to humans. Judging from these cases, other species of coronaviruses that use animals as hosts have the potential to evolve into mutant viruses that infect humans.

[0004] SARS-CoV-2 was first reported at the end of 2019. Compared to SARS-CoV, which originated in China in 2002, SARS-CoV has a higher severity, but SARS-CoV-2 has a higher transmissibility due to a mutation in the spike protein, which is the cell receptor binding site of SARS-CoV-2. This has resulted in a global pandemic. Common signs of infection include respiratory symptoms, fever, cough, shortness of breath, and difficulty breathing. In more severe cases, the infection can cause pneumonia, severe acute respiratory syndrome, kidney failure, and even death.

[0005] Currently, several countries and organizations are making efforts to develop a vaccine to eradicate the COVID-19 pandemic. The types of preventive vaccines are classified into inactivated vaccines, attenuated vaccines, protein subunit vaccines, vaccines based on viral vectors, DNA vaccines, and mRNA vaccines according to the production method. Inactivated vaccines and live attenuated vaccines, which have traditionally been widely used as vaccine formulations, have the advantage of simple production methods, but inactivated vaccines do not induce high levels of immune responses, and live attenuated vaccines have the risk of reacquiring pathogenicity through mutation. The problem is that protein subunit vaccines do not induce much immune response, so they must be used together with immune enhancers.

[0006] With the development of biotechnology, vaccine formulations based on new platforms have begun to be developed, one of which is the viral vector-based vaccine. Vector-based vaccines activate immune responses by delivering vaccine antigen genes to human cells with high efficiency to produce antigen proteins in the body itself. Viral vector-based vaccines are safe and induce high levels of immune responses, so efficient immunization is possible with only a single dose without the need for repeated administration. Viral vector-based vaccines also have the structural advantage of efficiently inducing virus-based antibody production as well as cytotoxic T cell immunity. Many viruses have been studied as gene vectors, and among them, adenoviruses are widely used in the field of gene therapy because they are easy to manipulate and their safety has been verified through many studies. Adenoviruses can deliver relatively large antigens (9-35 kb), making them highly competitive compared to other viral vectors.

[0007] Adenoviruses are linear, double-stranded DNA viruses with a diameter of 70-90 nm, non-enveloped, and have an icosahedral capsid formed by 240 hexons, 12 pentons, and fibers extending from each vertex of the icosahedron. These hexons, pentons, and fibers determine the major adenovirus antigens and its serotype. The adenovirus genome is approximately 30-45 kb in size and has four early regions (E1, E2, E3, and E4) and five late regions (L1-L5). Adenoviruses are good candidates as recombinant vectors for vaccine production because they are highly infectious and many of them are not pathogenic. In addition, adenovirus vectors can efficiently translate large genes and prolong immune responses in animals. Adenovirus vectors are primarily used in a form in which the E1 gene, which is responsible for autonomous replication, has been removed. Since adenovirus vectors with the E1 gene removed are unable to replicate autonomously in human cells, they are safe because they do not cause pathogenicity in the body.

[0008] Ad5 / 35 vector is a form in which the knob gene of non-replicating adenovirus serotype 5 (Ad5) is replaced with the knob gene of adenovirus serotype 35 (Ad35). This modification allows Ad5 / 35 vector to be introduced into cells via CD46 receptor, which is highly expressed in human immune cells, rather than the existing Ad5 cell receptor. That is, Ad5 / 35 can be efficiently introduced into human antigen-presenting cells, and thus can be an efficient vaccine platform for maximizing antigen production. Furthermore, Ad5 / 35 vector has a significantly lower risk of inducing hepatotoxicity compared with Ad5 vector. In fact, the present inventors have confirmed in preclinical and clinical trials that a high level of immune response is induced when cancer antigen genes are delivered to cancer patients using adenovirus vectors. Furthermore, it has been confirmed that there are no problems with stability and toxicity when administered to a large number of patients in clinical trials.

[0009] A consideration in the preparation of vaccine candidates is the stability of the antigens to be expressed. It is important that large amounts of vaccine antigens are secreted, but even if they are expressed, they cannot activate immune cells efficiently if they are degraded early and quickly. Therefore, it is important to maintain the antigens stable over long periods of time.

[0010] Therefore, in order to develop a vaccine for preventing or treating coronavirus disease-19 (COVID-19), the present inventors prepared a prophylactic or therapeutic vaccine by introducing into a vector a polynucleotide in which the cleavage site located between the S1 gene and the S2 gene encoding the spike protein, which plays an important role in cell infection by SARS-CoV-2, is replaced with a linker sequence. The present inventors then completed the present invention by confirming that the vaccine has high antibody production and T cell reactivity against SARS-CoV-2. Summary of the Invention

[0011] One object of the present invention is to provide a novel coronavirus recombinant spike protein, a polynucleotide encoding same, a vector comprising said polynucleotide, and a vaccine for preventing or treating coronavirus infection comprising said vector.

[0012] To achieve the above objective, the present invention provides a recombinant spike protein in which the cleavage site between the S1 gene and the S2 gene of the coronavirus spike protein has been removed and a linker sequence has been introduced.

[0013] The present invention also provides a recombinant spike protein in which the cleavage site between the S1 and S2 genes of the SARS-CoV-2 spike protein has been removed and a linker sequence has been introduced.

[0014] The present invention also provides a polynucleotide encoding a recombinant spike protein in which the cleavage site between the S1 and S2 genes of the coronavirus spike protein has been removed and a linker sequence has been introduced.

[0015] The present invention also provides a polynucleotide encoding a recombinant spike protein of SARS-CoV-2, in which the cleavage site between the S1 and S2 genes of the spike protein has been removed and a linker sequence has been introduced.

[0016] The present invention also provides a vector comprising a polynucleotide encoding a recombinant spike protein in which the cleavage site between the S1 and S2 genes of the coronavirus spike protein has been removed and a linker sequence has been introduced.

[0017] The present invention also provides a vector comprising a polynucleotide encoding a recombinant spike protein in which the cleavage site between the S1 and S2 genes of the SARS-CoV-2 spike protein has been removed and a linker sequence has been introduced.

[0018] The present invention also provides a vector characterized by introducing into an adenovirus a polynucleotide encoding a recombinant spike protein in which the cleavage site between the S1 and S2 genes of the coronavirus spike protein has been removed and a linker sequence has been introduced.

[0019] The present invention also provides a vector comprising a polynucleotide encoding a recombinant spike protein in which the cleavage site between the S1 and S2 genes of the coronavirus spike protein has been removed and a linker sequence has been introduced into an Ad5 / 35 virus.

[0020] The present invention also provides a vector characterized by introducing a polynucleotide encoding a recombinant spike protein in which the cleavage site between the S1 and S2 genes of the SARS-CoV-2 spike protein has been removed and a linker sequence has been introduced into an Ad5 / 35 virus.

[0021] The present invention also provides a vaccine for preventing or treating a coronavirus infection comprising a vector containing a polynucleotide of the S1 and S2 genes of the coronavirus spike protein.

[0022] The present invention also provides a vaccine for preventing or treating coronavirus infection comprising a vector containing polynucleotides of the S1 and S2 genes of the SARS-CoV-2 spike protein.

[0023] The present invention also provides a vaccine for preventing or treating coronavirus infection, characterized by introducing polynucleotides of the S1 and S2 genes of the SARS-CoV-2 spike protein into an adenovirus.

[0024] The present invention also provides a vaccine for preventing or treating coronavirus infection, characterized by introducing polynucleotides of the S1 and S2 genes of the SARS-CoV-2 spike protein into the Ad5 / 35 virus.

[0025] Furthermore, the present invention provides a vaccine for preventing or treating coronavirus disease 19 (COVID-19), comprising a vector characterized by introducing into an Ad5 / 35 virus a polynucleotide in which the cleavage site between the S1 and S2 genes of the SARS-CoV-2 spike protein has been removed and a linker sequence has been introduced. Effect of the Invention

[0026] The present invention relates to a novel coronavirus recombinant spike protein, a polynucleotide encoding the same, a vector comprising said polynucleotide, and a vaccine comprising said vector for preventing or treating coronavirus infection. The coronavirus recombinant spike protein of the present invention is stable, and therefore is not easily degraded in cells, and efficiently activates immune cells, resulting in high antibody production and T cell reactivity. It has been confirmed that the vector of the present invention exhibits a high antigen expression level, and therefore has high antibody production and T cell reactivity, has a long antibody production and expression period, and does not exhibit liver toxicity. Therefore, the vector of the present invention can be used to serve as a vaccine for preventing or treating coronavirus infection. [Brief description of the drawings]

[0027] [Figure 1] FIG. 1 is a schematic diagram showing a vaccine for preventing or treating COVID-19 according to the present invention, in which the E3 gene region, which is the receptor binding site, is replaced with the fiber gene of adenovirus serotype 35 based on adenovirus serotype 5, and spike protein, which plays an important role in cell infection by SARS-CoV-2, is loaded as an antigen. [Diagram 2]FIG. 1 is a schematic diagram showing that the vaccine for preventing or treating COVID-19 of the present invention comprising a recombinant spike protein, in which the cleavage site of the gene encoding the spike protein is replaced with a linker sequence as an antigen, has structural stability and high antigen expression, and a good ability to produce neutralizing antibodies in antibody-producing B cells. [Diagram 3] FIG. 1 is a schematic diagram showing the mechanism of action of the vaccine for preventing or treating coronavirus disease-19 of the present invention. [Figure 4] FIG. 1 is a schematic diagram showing a control expression vector prepared using the adenovirus type 5 (Ad5) genome. [Diagram 5] FIG. 1 is a schematic diagram showing adenoviral vectors #1 to #4 loaded with a spike protein antigen, which is an antigen expression gene. [Figure 6] FIG. 1 shows the expression levels of antigens when adenovirus vectors #1 to #4 loaded with spike protein antigens, which are antigen expression genes, are introduced into cultured cell lines. [Figure 7] FIG. 1 shows the expression levels of antigens secreted from cells when adenovirus vectors #1 to #4 loaded with spike protein antigens, which are antigen expression genes, are introduced into cultured cell lines. [Figure 8] FIG. 1 shows the amount of antibody production after injecting mice with adenovirus vectors #1 to #4 loaded with a spike protein antigen, which is an antigen expression gene. [Figure 9] FIG. 13 shows the amount of antigen-specific antibodies present in the blood after mice were injected with adenovirus vector #3 loaded with spike protein antigen, which is an antigen expression gene. [Figure 10] FIG. 13 shows the amount of antigen-specific antibodies present in the blood after injecting monkeys with adenovirus vector #3 loaded with spike protein antigen, which is an antigen-expressing gene. [Figure 11] FIG. 13 shows the concentration of neutralizing antibodies after injecting adenovirus vector #3 loaded with a spike protein antigen, which is an antigen expression gene, into monkeys. [Figure 12]FIG. 1 shows the levels of liver toxicity in the blood after monkeys were injected with adenoviral vectors #1 to #4 loaded with a spike protein antigen, which is an antigen expression gene. [Figure 13] FIG. 1 shows T cell reactivity in spleen cells and peripheral blood mononuclear cells (PBMCs) after mice were injected with adenovirus vectors #1 to #3 loaded with spike protein antigen, which is an antigen expression gene. [Figure 14] FIG. 1 shows T cell reactivity in peripheral blood mononuclear cells (PBMCs) after injection of adenoviral vector #3 loaded with spike protein antigen, an antigen expression gene, into primates. [Figure 15] FIG. 11 confirms that similar levels of neutralizing antibodies were produced when mice were injected with Vector #3 expressing a spike antigen prepared in Example 3 or a vector in which the E4 gene, among the adenoviral genes of Vector #3, was rearranged into the E1 region of the viral genome. [Figure 16] FIG. 11 confirms that similar levels of neutralizing antibodies were produced when spike antigen-expressing vector #3 prepared in Example 3 or a vector in which the E4 gene, among the adenoviral genes of vector #3, has been rearranged into the E1 region of the viral genome was injected into monkeys. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] The present invention will be described in detail below.

[0029] The present invention provides a recombinant spike protein in which the cleavage site between the S1 and S2 genes of the coronavirus spike protein has been removed and a linker sequence has been introduced.

[0030] The coronavirus is characterized in that it is SARS-CoV, MERS-CoV or SARS-CoV-2, which belongs to the Betacoronavirus.

[0031] The genome homology between SARS-CoV-2 and SARS-CoV is 79.6%, and between SARS-CoV-2 and MERS-CoV it reaches 50%. In particular, the homology of the spike protein, which is the target of the present invention, is similar at 35% between SARS-CoV-2 and MERS-CoV, but the spike protein homology between SARS-CoV-2 and SARS-CoV is very high at 76%.

[0032] The present invention also provides a recombinant spike protein in which the cleavage site between the S1 and S2 genes of the SARS-CoV-2 spike protein has been removed and a linker sequence has been introduced.

[0033] In a vaccine, it is important that antigens are secreted in large quantities, but even if they are expressed in large quantities, if they are degraded early, they will not stimulate immune cells and will not induce sufficient immune responses, so it is very important that the expressed antigens have high stability. The recombinant spike protein of the present invention, in which the linker sequence is linked between S1 and S2, has high antigen expression and improved stability, and thus has excellent ability to generate neutralizing antibodies and induce T cell reactivity.

[0034] The term "immune response" may include, but is not always limited to, humoral and cellular immune responses, such as CD4+ or CD8+ cell activation.

[0035] Spike protein antigens linked by linker sequences are characterized by increased antigen expression.

[0036] Spike protein (S protein) covers the surface of coronavirus particles. It is a spike-like protein used by coronaviruses to enter human cells and consists of S1 and S2 subunits. Coronaviruses infect the human body in such a way that the spike protein binds to the angiotensin-converting enzyme 2 (ACE-2) receptor of human cells, penetrates into the cells, inserts its genetic material (RNA) into the cells, and self-replicates.

[0037] The recombinant protein is characterized by the removal of the cleavage site between the S1 and S2 genes, which are subunits of the spike protein.

[0038] This protein is characterized in that the cleavage site between the S1 and S2 genes has been removed and they are connected by a linker sequence.

[0039] The linker will look like this: (GGGGS)n (SEQ ID NO:31) (wherein n is an integer of 1 to 5).

[0040] The domains constituting each protein have an optimized distance (length of the linker sequence) for interaction due to their unique characteristics, and the structural stability of the protein is represented by the length of the linker sequence. Therefore, n of the protein may be preferably 1 to 3, most preferably 1.

[0041] If n is greater than 4, numerous repeat sequences are generated and the linker sequence may be deleted by unintended homologous recombination, reducing protein stability.

[0042] Spike protein antigens linked by linker sequences are characterized by increased stability. The linker sequence is characterized in that it consists of SEQ ID NO:19 or SEQ ID NO:20.

[0043] All coronaviruses have in common the spike protein, which is composed of S1 and S2 subunits, and therefore any spike protein of a coronavirus can be used.

[0044] All spike proteins in which the cleavage site between the S1 and S2 genes of the spike protein has been removed and linked by a linker belong to the present invention.

[0045] Coronaviruses include 229E, OC43, NL63 and HKU1, which cause the common cold, and SARS-CoV, MERS-CoV and SARS-CoV-2, which cause severe pneumonia. All of the spike proteins of these coronaviruses can be used.

[0046] Preferably, it is a recombinant spike protein in which the cleavage site between the S1 and S2 genes of the spike protein of SARS-CoV-2 has been removed and a linker sequence has been introduced.

[0047] The recombinant spike protein may further contain an adjuvant to improve immunogenicity, such as GM-CSF, IL-17, IFN-Ng, IL-15, IL-5, JNK, NFkB, NKKKKLIGAND, PD1 / 2, NKKKKG2B, NKG2C, NKKKKG2E, NKKKK2F, TAPAPAP2 and its functional fragments, E-selectin, IL-α, IL-6, INF-γ, lymphotoxin α, hGH-1, MIPPP1, IL-7, IL-8, APP, IRARAK, IkB, KI It may be at least one selected from the group consisting of LILUX, TRAIL-1, IL-1, AIR, and ICAM-1, a functional fragment of ICAM-1, TAP2, CD40, TAP1, TRAILrecDRC5, CD34, CD40L, DR3, GlyCAM1, p55, CD2, Ox40, TRAF6, DR4, ICAM3, DR5, MyD88, p65Rel, pl5095, p38, CPG and TLR.

[0048] The adjuvant can be linked to the C-terminus of the recombinant spike protein.

[0049] The present invention also provides a polynucleotide encoding a recombinant spike protein in which the cleavage site between the S1 and S2 genes of the coronavirus spike protein has been removed and a linker sequence has been introduced.

[0050] The coronavirus is characterized as being SARS-CoV, MERS-CoV or SARS-CoV-2.

[0051] The present invention also provides a polynucleotide encoding a recombinant spike protein of SARS-CoV-2, in which the cleavage site between the S1 and S2 genes of the spike protein has been removed and a linker sequence has been introduced.

[0052] The linker is characterized in that it consists of 9 to 45 nucleotides, preferably 9 to 21 nucleotides, and more preferably 15 nucleotides.

[0053] Polynucleotides encoding spike proteins linked by linker sequences are characterized by increased stability.

[0054] Polynucleotides encoding spike proteins linked by linker sequences are characterized by increased antigen expression.

[0055] The linker sequence is characterized in that it consists of SEQ ID NO:11 or SEQ ID NO:15.

[0056] The polynucleotide may be SEQ ID NO:12 or SEQ ID NO:16.

[0057] The present invention also provides a vector comprising a polynucleotide encoding a recombinant spike protein in which the cleavage site between the S1 and S2 genes of the coronavirus spike protein has been removed and a linker sequence has been introduced.

[0058] The coronavirus is characterized as being SARS-CoV, MERS-CoV or SARS-CoV-2.

[0059] The present invention also provides a vector comprising a polynucleotide encoding a recombinant spike protein in which the cleavage site between the S1 and S2 genes of the SARS-CoV-2 spike protein has been removed and a linker sequence has been introduced.

[0060] The present invention also provides a vector characterized by introducing into an adenovirus a polynucleotide encoding a recombinant spike protein in which the cleavage site between the S1 and S2 genes of the coronavirus spike protein has been removed and a linker sequence has been introduced.

[0061] The present invention also provides a vector characterized by introducing into an adenovirus a polynucleotide encoding a recombinant spike protein in which the cleavage site between the S1 and S2 genes of the SARS-CoV-2 spike protein has been removed and a linker sequence has been introduced.

[0062] The vector prepared by introducing a polynucleotide encoding a recombinant spike protein in which the cleavage site between the S1 and S2 genes of the SARS-CoV-2 spike protein has been removed and a linker sequence has been introduced into an adenovirus may be SEQ ID NO: 27 or SEQ ID NO: 29.

[0063] The present invention also provides a vector comprising a polynucleotide encoding a recombinant spike protein in which the cleavage site between the S1 and S2 genes of the coronavirus spike protein has been removed and a linker sequence has been introduced into an Ad5 / 35 virus.

[0064] The present invention also provides a vector characterized by introducing a polynucleotide encoding a recombinant spike protein in which the cleavage site between the S1 and S2 genes of the SARS-CoV-2 spike protein has been removed and a linker sequence has been introduced into an Ad5 / 35 virus.

[0065] In the present invention, the term "vector" refers to a transporter that carries a cloned gene (or other piece of cloned DNA) and transports it to a target cell.

[0066] The vector may be any one selected from the group consisting of a plasmid and a virus.

[0067] Specific examples of plasmid DNA include commercially available plasmids such as pCMV3, pET28a and pET. Other examples of plasmids that can be used in the present invention include E. coli derived plasmids (pCMV3, pET28a, pET, pGEX, pQE, pDEST and pCOLD), Bacillus subtilis derived plasmids (pUB110 and pTP5) and yeast derived plasmids (YEp13, YEp24 and YCp50). These plasmids show different amounts of protein expression and modification depending on the host cell, so the most suitable host cell for the purpose can be selected and used.

[0068] The virus can be any known virus that can be used as a vector, and can be any one selected from the group consisting of adenovirus, retrovirus, lentivirus, adeno-associated virus (AAV), modified vaccinia virus Ankara (MVA), herpes simplex virus and baculovirus, but is not always limited thereto.According to a particular embodiment of the present invention, the virus is preferably adenovirus.

[0069] The term "adenovirus vector" as used herein refers to an adenovirus that has been engineered such that the adenovirus genome has a nucleic acid sequence that is non-natural with respect to the adenovirus genome. Thus, as used herein, a "recombinant adenovirus vector" typically comprises an expression cassette that contains the adenovirus genome and at least one foreign nucleic acid encoding a required protein (e.g., a recombinant spike protein in which the cleavage site between S1 and S2 of the SARS-CoV-2 spike protein is replaced with a linker sequence).

[0070] Adenoviral vectors preferably contain at least a portion of each terminal repeat sequence required to support viral DNA replication, preferably at least about 90% of the complete inverted terminal repeat (ITR) sequence, and the DNA required to encapsidate the genome into the viral capsid. Adenoviruses from various sources can be used as the source of viral genome for adenoviral vectors. Human adenoviruses such as subgroup A (e.g., serotypes 12, 18 and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, etc.), subgroup C (e.g., serotypes 1, 2, 5 and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, etc.), subgroup E (e.g., serotype 4), subgroup F (e.g., serotypes 40 and 41), etc. are preferred. Preferably, the adenoviral vector is a vector of human subgroup C, in particular serotype 2, more preferably serotype 5.

[0071] Adenoviral vectors can be replication-competent vectors. Typically, adenoviral vectors are replication-deficient vectors in host cells. The term "replication-deficient" means that adenoviral vectors have a deficiency in one or more gene functions or regions (e.g., E1, E3 or E4 regions) of the adenoviral genome for replication, and thus the vector maintains some low level of replication or does not replicate in normal host cells, particularly human cells infected by adenoviral vectors. Replication-deficient adenoviral vectors ensure the safety of vaccines. In one embodiment of the present invention, the adenoviral vector is a vector in which E1 or E3 or both are deleted. Genetic deficiency is defined as a mutation or deletion that completely removes or impairs the function of a gene, for example, thus the function of gene product is reduced to at least about 1 / 2, 1 / 5, 1 / 10, 1 / 20 or less of the function of natural gene. The resulting replication-defective adenoviral vector can accommodate one or more exogenous nucleic acids at appropriate sites in the adenoviral genome for the expression of one or more desired proteins while retaining the ability to be packaged in adenoviral capsid.To generate high titer viral vector for stock solution, replication-defective adenoviral vector is typically produced in a complementation cell line such as HEK293 or HEK293R cell, which provides gene functions that are not present in replication-defective adenoviral vector.

[0072] The adenovirus may be adenovirus serotype 2 (Ad2), adenovirus serotype 4 (Ad4), adenovirus serotype 5 (Ad5), adenovirus serotype 11 (Ad11), adenovirus serotype 26 (Ad26), adenovirus serotype 35 (Ad35), chimpanzee adenovirus serotype 68 (ChAd68), poultry adenovirus serotype 9 (FAd9) or porcine adenovirus serotype 3 (PAd3).

[0073] The adenovirus may have a modified form based on Ad5.

[0074] The adenovirus may be an adenovirus in which the knob gene of adenovirus serotype 5 (Ad5) has been replaced with the knob gene of adenovirus serotype 35 (Ad35) (Ad5 / 35).

[0075] Adenoviruses are characterized by deletions of the E1 and E3 genes.

[0076] The adenovirus may have a form in which a part or the whole of the E4 gene is deleted, and in addition to deleting the E1 and E3 genes, the E4 gene is rearranged into the E1 region.

[0077] Adenovirus is characterized in that it is introduced into cells via the CD46 receptor, which is highly expressed in human immune cells.

[0078] Adenoviruses are characterized by their low efficacy in inducing hepatotoxicity.

[0079] Adenoviruses are characterized by a high degree of antigen expression.

[0080] The present invention also provides a vaccine for preventing or treating a coronavirus infection comprising a vector containing a polynucleotide of the S1 and S2 genes of the coronavirus spike protein.

[0081] The coronavirus may be any one selected from the group consisting of SARS-CoV, MERS-CoV and SARS-CoV-2.

[0082] The vector can be any known virus that can be used as a vector, and can be any one selected from the group consisting of adenovirus, retrovirus, lentivirus, adeno-associated virus (AAV) and modified vaccinia virus Ankara (MVA), but is not always limited thereto.According to a particular embodiment of the present invention, the virus is preferably an adenovirus.

[0083] The adenovirus may be adenovirus serotype 2 (Ad2), adenovirus serotype 4 (Ad4), adenovirus serotype 5 (Ad5), adenovirus serotype 11 (Ad11), adenovirus serotype 26 (Ad26), adenovirus serotype 35 (Ad35), chimpanzee adenovirus serotype 68 (ChAd68), poultry adenovirus serotype 9 (FAd9) or porcine adenovirus serotype 3 (PAd3).

[0084] The adenovirus may have a modified form based on Ad5.

[0085] The adenovirus may be an adenovirus in which the knob gene of adenovirus serotype 5 (Ad5) has been replaced with the knob gene of adenovirus serotype 35 (Ad35) (Ad5 / 35).

[0086] Adenoviruses are characterized by deletions of the E1 and E3 genes.

[0087] The adenovirus may have a form in which a part or the whole of the E4 gene is deleted, and in addition to deleting the E1 and E3 genes, the E4 gene is rearranged into the E1 region.

[0088] Adenovirus is characterized in that it is introduced into cells via the CD46 receptor, which is highly expressed in human immune cells.

[0089] The present invention also provides a vaccine for preventing or treating coronavirus infection comprising a vector containing polynucleotides of the S1 and S2 genes of the SARS-CoV-2 spike protein.

[0090] The present invention also provides a vaccine for preventing or treating coronavirus infection, characterized by introducing polynucleotides of the S1 and S2 genes of the SARS-CoV-2 spike protein into an adenovirus.

[0091] The present invention also provides a vaccine for preventing or treating coronavirus infection, characterized by introducing polynucleotides of the S1 and S2 genes of the SARS-CoV-2 spike protein into the Ad5 / 35 virus.

[0092] Furthermore, the vaccine of the present invention can be prepared as an oral or parenteral formulation and can be administered by intradermal, intramuscular or intranasal routes, preferably by the intramuscular route.

[0093] The vaccine of the present invention is characterized in that the cleavage site between S1 and S2 is deleted.

[0094] The vaccine is characterized in that the cleavage site between S1 and S2 of the spike protein has been removed and joined with a linker sequence.

[0095] The linker is characterized in that it consists of 9 to 45 nucleotides, preferably 9 to 21 nucleotides, and more preferably 15 nucleotides.

[0096] Additionally, the present invention provides methods for preventing or treating a coronavirus infection, including coronavirus disease-19, severe acute respiratory syndrome (SARS), or Middle East respiratory syndrome (MERS), by administering the vaccine to a subject.

[0097] In the present invention, the term "subject" refers to all animals, including humans, that can be infected with coronavirus. By administering the vaccine of the present invention to a subject, the above-mentioned disease can be effectively prevented. For example, the vaccine of the present invention can prevent humans from being infected with coronavirus.

[0098] In the present invention, the term "prevention" refers to any action that inhibits or delays the onset of a coronavirus infection by administering a vaccine.

[0099] In the present invention, the term "treatment" means any action of eliminating the coronavirus or alleviating the symptoms caused by the coronavirus by administering a vaccine.

[0100] The vaccine of the present invention is administered in a pharmacologic effective dose. The term "pharmacologic effective dose" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment or improvement. The effective dose level depends on factors including the type and severity of the subject, age, gender, type of virus infected, activity of the drug, susceptibility to the drug, time of administration, route of administration, rate of excretion, duration of treatment, concomitant medications, and other factors well known in the medical community. The vaccine of the present invention can be administered alone or in combination with other therapeutic agents. In the case of concomitant administration, administration can be sequential or simultaneous. And the vaccine can be administered once or multiple times. It is important to administer an amount that can obtain maximum effect at the minimum amount without side effects, taking into account all of the above factors, and this can be easily determined by those skilled in the art.

[0101] The term "effective amount" refers to a dose sufficient to produce a desired therapeutic effect in the treated subject, for example, sufficient to generate or induce an immune response against a pathogen or antigen in the recipient. The effective amount may vary depending on various reasons, such as the route of administration, the frequency of administration, the weight and species of the individual receiving the drug, and the purpose of administration. Those skilled in the art can determine the dosage for each case based on the disclosure of the present specification, established methods, and their own experience. For example, in a certain embodiment of the present invention, the recombinant adenovirus vector of the present invention is administered in a dose of 1x10 9 ~1x10 12 The vaccine may be administered in a dose of 1x10 viral particles (VP), for example 1x10 9 , 1x10 10 , 1x10 11 , or 1x10 12 The VP may be administered in a dose of 100 mg / kg / day.

[0102] In a specific embodiment of the present invention, the inventors constructed a vector by introducing a polynucleotide in which the cleavage site between S1 and S2 of a polynucleotide encoding the spike protein of SARS-CoV-2, which belongs to coronavirus, was replaced with a linker sequence into an adenovirus in which the E1 and E3 genes were deleted and the fiber, which is the cell receptor binding site of adenovirus serotype 5, was replaced with the fiber of serotype 35 (see FIG. 4), and confirmed that the vector had high antigen expression, antibody production and T cell reactivity, a long antibody production expression period, and no liver toxicity (see FIGS. 6, 7, 9, 12, 13, and 14). Furthermore, a vaccine prepared by loading an antigen into a vector in which a part or all of the E4 gene was deleted and in which the E1 gene and E3 gene were deleted and the E4 gene was rearranged to the E1 region was administered to mice and monkeys. As a result, it was confirmed that the production amount of neutralizing antibodies was similar to that of a vaccine prepared by loading an antigen into an Ad5 / 35 vector (see FIGS. 15 and 16).

[0103] The coronavirus recombinant spike protein of the present invention is stable, and therefore is not easily degraded in cells, and can efficiently activate immune cells, resulting in high antibody production and T cell reactivity. It has been confirmed that the vector of the present invention exhibits high antigen expression levels, and therefore has high antibody production and T cell reactivity, has a long antibody production and expression period, and does not exhibit liver toxicity. Therefore, the vector of the present invention can be used to serve as a vaccine for preventing or treating coronavirus infection.

[0104] Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples.

[0105] However, the following examples and experimental examples are merely intended to illustrate the present invention, and the contents of the present invention are not limited thereto. EXAMPLES

[0106] Construction of control expression vector (plasmid vector #1) A plasmid containing the adenovirus sequence (SEQ ID NO:21) without the spike protein antigen was constructed using the genome of adenovirus type 5 (Ad5), designated pAdk35F (SEQ ID NO:1, 34,030 bp). pAdk35F contains an adenovirus in which the E1 and E3 genes have been deleted and the fiber, which is the cell receptor binding site of adenovirus serotype 5, has been replaced with the fiber of serotype 35. The E1 gene deletion site of pAdk35F is loaded with a CMV (cytomegalovirus) promoter, a tetracycline operator (TetO), a restriction enzyme SwaI site, and an SV40 polyA sequence.

[0107] As shown in Figure 4, the extracellular domain of the spike protein antigen gene was amplified using the pCMV3-SARS-CoV-2 (spike ORF) vector (SinoBiological, Cat. No. VG40589-UT, SEQ ID NO: 2, 9,858 bp) loaded with the polynucleotide encoding the spike protein, which is the antigen expression gene, as a template, using the primer set for amplifying the spike protein antigen listed in Table 1 below, and then the control expression vector, plasmid vector #1 (SEQ ID NO: 22), was constructed by in-fusion cloning ligation with pAdk35F vector digested with SwaI (Clontech, Cat. No. 639648). The region encoding the spike protein in the plasmid vector #1 is represented by SEQ ID NO: 3, and the nucleotide sequence of the adenovirus into which the spike protein is introduced is represented by SEQ ID NO: 23.

[0108] [Table 1] EXAMPLES

[0109] Construction of an improved spike antigen expression vector <2-1> Construction of an improved spike antigen expression vector (plasmid vector #2) The cleavage sites of domains S1 and S2 of the spike protein antigen of the pCMV3-SARS-CoV-2 (spike ORF) vector used as a template for polymerase chain reaction in Example 1 (SEQ ID NO: 6, amino acid sequence: TNSPRRAR (SEQ ID NO:32) ) was synthesized by site-directed mutagenesis using the primer sets listed in Table 2 below, TILR (Thr-Ile- Leu - Arg )array( SEQ ID NO:33 ) to prepare the pCMV3-SARS-CoV-2(spike IL) vector as shown in Figure 5.

[0110] The vector replaced with the TILR sequence was then amplified using the same primer set for amplifying the spike antigen listed in Table 1 as the plasmid vector #1, and the improved spike antigen expression vector, plasmid vector #2 (SEQ ID NO:24), was then constructed by in-fusion cloning ligation with pAdk35F vector digested with SwaI (Clontech, Cat. No. 639648). The region encoding the spike protein in the plasmid vector #2 is represented by SEQ ID NO:8, and the nucleotide sequence of the adenovirus into which the spike protein is introduced is represented by SEQ ID NO:25.

[0111] [Table 2]

[0112] <2-2> Construction of an improved spike antigen expression vector (plasmid vector #3; pAdCLD-CoV19 vector) The cleavage sites of domains S1 and S2 of the spike protein antigen of the pCMV3-SARS-CoV-2 (spike ORF) vector used as a template for polymerase chain reaction in Example 1 (SEQ ID NO: 6, amino acid sequence: TNSPRRAR (SEQ ID NO:32)) was converted to the TGGGGSR linker sequence ( SEQ ID NO:19 ) to prepare pCMV3-SARS-CoV-2 (spike GGGGS) vector. The vector replaced with the TGGGGSR sequence was then amplified using the same primer set for amplifying the spike antigen as in plasmid vector #1, listed in Table 1, and the improved spike antigen expression vector, plasmid vector #3 (pAdCLD-CoV19 vector) (SEQ ID NO: 26), was then constructed by in-fusion cloning ligation with pAdk35F vector digested with SwaI (Clontech, Cat. No. 639648). The region encoding the spike protein in plasmid vector #3 is represented by SEQ ID NO: 12, and the nucleotide sequence of the adenovirus into which the spike protein is introduced is represented by SEQ ID NO: 27.

[0113] [Table 3]

[0114] <2-3> Construction of improved spike antigen expression vector (plasmid vector #4) The cleavage sites of domains S1 and S2 of the spike protein antigen of the pCMV3-SARS-CoV-2 (spike ORF) vector used as a template for polymerase chain reaction in Example 1 (amino acid sequence: TNSPRRAR (SEQ ID NO:32) ) was synthesized by site-directed mutagenesis using the primer sets listed in Table 4 below to produce the sequence TGGGGSGGGGSGGGGSR ( SEQ ID NO:34) to prepare pCMV3-SARS-CoV-2 (spike GGGGSx3) vector. The vector replaced with the TGGGGSGGGGSGGGGSR sequence was then amplified using the same primer set for amplifying the spike antigen as in plasmid vector #1, and an improved spike antigen expression vector, plasmid vector #4 (SEQ ID NO: 28), was constructed by in-fusion cloning ligation with pAdk35F vector digested with SwaI (Clontech, Cat. No. 639648). The region encoding the spike protein in plasmid vector #4 is represented by SEQ ID NO: 16, and the nucleotide sequence of the adenovirus into which the spike protein is introduced is represented by SEQ ID NO: 29.

[0115] [Table 4] EXAMPLES

[0116] Construction of spike antigen-loaded adenovirus vectors (vectors #1–#4) Adenoviral vectors (vectors #1-#4) were prepared from the vectors in Examples 1 and 2 above using HEK293R cells. Initial adenoviral vectors were produced by transfecting approximately 80% confluent HEK293R cells in a T25 plate with 12.5 μg of each vector along with 25 μl of Lipofectamine 2000 (ThermoFisher, Cat. No. 11668027). Each initial adenovirus was then transfected into 4x10 7 The plasmid was amplified by infecting 20 T175 flasks containing HEK293R cells.

[0117] The amplified adenovirus vectors #1 to #4 were purified by the first (1.2 g / ml CsCl + 1.4 g / ml CsCl, 32,000 RPM, 90 min) and second (1.35 g / ml CsCl, 32,000 RPM, 18 h) cesium chloride (CsCl) density gradient centrifugation. The first adenovirus was produced by dialysis (20 mM Tris-HCl, 25 mM sodium chloride, 2.5% glycerol). EXAMPLES

[0118] Comparison of antigen expression levels of antigen expression vectors in vitro <4-1> Comparison of intracellular antigen expression by antigen expression vector injection To compare the intracellular antigen expression levels of vectors #1 to #4 prepared in Example 3, the antigen expression levels in the THP-1 cell line derived from mononuclear cells were measured by flow cytometry (FACS).

[0119] Specifically, for flow cytometry, THP-1 cells were plated at 2.5x10 per well. 5 The cells were inoculated with each vector at a multiplicity of infection (MOI) of 10 and then cultured for 24 hours. The cells in each well were then transferred to a 1.5 ml tube, 500 μl of FACS buffer was added, and centrifuged (5000 rpm, 3 min, 4° C.). After removing the supernatant, 50 μl of a viability indicator dye (eFluor™ 450, ebioscience, catalog number 65-0863-14) diluted 1:1000 was added to each well, and surface staining was performed at 4° C. for 30 min.

[0120] To detect intracellularly expressed antigens, Cell Fixation / Permeabilization Concentrate (ebioscience, Catalog No. 00-5123-43) was diluted 1:4 in Cell Fixation / Permeabilization Diluent (ebioscience, Catalog No. 00-5223-56) and 100 μl of the solution was added to each well followed by cell fixation / permeabilization at 4° C. for 30 minutes. Spike antigen staining was performed with a solution of SARS-CoV-2 spike protein antibody (GeneTex, catalog number GTX632604) diluted 1:2500 in permeabilization buffer (ebioscience, catalog number 00-8333-56) (antibody concentration: 0.4 μg / ml) for 1 h at 4 °C, followed by washing and then antigen staining with a solution of secondary antibody APC goat anti-mouse IgG antibody (Biolegend, catalog number 405308) diluted 1:100 (antibody concentration: 2 μg / ml) (4 °C, 30 min). After the staining reaction, 300 μl of buffer was added per sample, and the degree of antigen expression was measured by flow cytometry (BD bioscience, LSR FORESA).

[0121] As a result, as shown in FIG. 6, it was confirmed that the adenoviral vector #3 prepared in Example 3 had the highest antigen expression level, which is the ability to produce protein.

[0122] <4-2> Comparison of extracellular antigen expression by antigen expression vector injection To compare the expression levels of extracellular antigens in vectors #1 to #4 prepared in Example 3, the antigen expression levels in mononuclear cell-derived THP-1 cells and muscle cell-derived RD cells were measured by Western blotting.

[0123] Specifically, the Western blotting method for measuring the expression level of the spike protein antigen excreted from cells is as follows. THP-1 and RD cells were cultured at 5x10 per well. 5The cells were distributed at a density of 1000 x 1000 cells into 6-well culture dishes, and then each vector was inoculated into the cells at an MOI of 50, followed by culturing for 24 hours. The culture medium was then harvested by centrifugation, concentrated using Microcon (Millipore, 50,000 MWCO, Cat. No. UFC805024), and the cultured cells were decomposed into protein units using RIPA buffer (ThermoFisher, Cat. No. 89901). The total amount of protein in the concentrated culture medium was measured using a BCA protein assay kit (ThermoFisher, Cat. No. 23225).

[0124] To confirm the presence of spike proteins (trimers, ≧250 kDa) in the concentrated culture media samples, samples (60 μg / well) were electrophoresed at 180 V using Bolt™ Bis-Tris 4-12% Plus gels (ThermoFisher, Cat. No. NW04120BOX).

[0125] The electrophoresed gel was transferred to a PVDF membrane using an iBlot 2 Dry Blotting System (Invitrogen, Cat. No. IB21001) for 10 min at 20 V. The membrane was then reacted with rabbit SARS-CoV-2 spike antibody (GeneTex, Cat. No. GTX135360) and goat anti-rabbit IgG H&L (HRP), respectively, to detect the trimeric spike protein of SARS-CoV-2 using ECL Prime Western Blotting Detection Reagent (Amersham, Cat. No. RPN2232).

[0126] As a result, as shown in FIG. 7, it was confirmed that the adenovirus vector #3 prepared in Example 3 had the highest antigen expression for producing and excreting protein. EXAMPLES

[0127] Comparison of antibody production in mice injected with antigen-expressing vectors <5-1> Comparison of antibody production in mice by injection of antigen expression vectors #1 to #4 Vectors #1 to #4 prepared in Example 3 were injected into mice, and the amounts of antibody production were compared.

[0128] Specifically, the spike antigen expression vectors (vectors #1 to #4) prepared in Example 3 were administered at 2 x 10 8 A dose of 1000 IFU / mouse was injected intramuscularly into 6-7 week old BALB / c mice. Approximately 300 μl of blood samples were collected by retro-orbital blood sampling 2-3 weeks after administration. Plasma was then separated by centrifugation (8000 rpm, 10 min, 20°C) and the amount of neutralizing antibodies present in the blood was measured by enzyme-linked immunosorbent assay (ELISA).

[0129] For enzyme-linked immunosorbent assay, 96-well plates were coated with spike protein (Acro Biosystems, Cat. No. SPN-C52H84) dissolved in PBSN (PBS 1L + sodium azide 0.01g) at 100ng / well, followed by cold reaction at 4°C for 16 hours. After coating the 96-well plates for 16 hours, the coating protein was removed, washed three times with PBS, and then 150μl of blocking buffer (PBSN+BSA 1%) was added to each well, followed by reaction (37°C, 90 minutes). During the reaction time, plasma was diluted 6400 times in dilution buffer (PBSN+0.1%BSA+0.05%Tween-20), and after the blocking reaction, the plate was washed three times with PBS, and then 50μl of diluted plasma sample was added to each well, followed by reaction (37°C, 3 hours). Upon completion of the reaction, the plate was washed three times with PBS, and 50 μl of secondary antibodies, GAM-IgG-HRP (Southernbiotech, Cat. No. 1030-05) and GAM-IgM-HRP (Southernbiotech, Cat. No. 1020-05), diluted 1000 times in dilution buffer, were added to each well, followed by reaction (37° C., 2 hours). The secondary antibody was then removed, the plate was washed five times with PBS, and 50 μl of color development reagent (TMB peroxidase substrate buffer, ROCKLAND, Cat. No. TMBE-1000) was added to each well, followed by color development for 15 minutes. Color development was stopped by adding 50 μl of 0.25 N HCl to each well, and the samples were analyzed at a wavelength of 450 nm using a microreader.

[0130] As a result, as shown in FIG. 8, it was found that the adenoviral vector #3 prepared in Example 3 produced the highest amount of antibody.

[0131] <5-2> Comparison of antibody production and expression period in mice by injection of antigen expression vector Furthermore, the adenovirus vectors (vectors #1 to #3) prepared in Example 3 were administered at a dose of 1 x 10 9 VP / mouse or 2x10 8VP / mouse was intramuscularly injected into 8-week-old BALB / c mice. Approximately 300 μl of blood samples were collected by retro-orbital blood sampling 2-4 weeks after administration. Plasma was then separated by centrifugation (8000 rpm, 10 min, 20°C), and the amount of antigen-specific antibodies present in the blood was measured by enzyme-linked immunosorbent assay (ELISA) as described in Example 5-1.

[0132] As a result, as shown in FIG. 9, it was found that the amount of antigen-specific antibodies present in the blood of mice injected with adenovirus vector #3 prepared in Example 3 was maintained at a higher level than that of vector #2 for more than four weeks. EXAMPLES

[0133] Comparison of antibody production and hepatotoxicity in monkeys following injection of antigen-expressing vectors <6-1> Comparison of antibody production in monkeys by injection of antigen expression vectors The amount of antibody production in monkeys after injection of the adenoviral vectors prepared in Example 3 (vectors #1 and #3) was compared.

[0134] Specifically, the adenoviral vectors #1 and #3 prepared in Example 3 were administered at 1x10 11 A dose of VP / monkey was injected intramuscularly into 6-7 week-old cynomolgus monkeys. Two and three weeks after immunization, blood samples were collected from the monkeys by intravenous blood sampling. Plasma was then separated by centrifugation (8000 rpm, 10 min, 20°C), and the amount of antibodies present in the blood was measured by enzyme-linked immunosorbent assay (ELISA) and pseudovirus neutralization assay.

[0135] For enzyme-linked immunosorbent assay, 96-well plates were coated with spike protein (Acro Biosystems, Cat. No. SPN-C52H84) dissolved in PBSN at 100ng / well, followed by cold reaction at 4°C for 16 hours. After coating the 96-well plates for 16 hours, the coating protein was removed, washed three times with PBS, and then 150μl of blocking buffer was added to each well, followed by reaction (37°C, 90 minutes). During the reaction time, plasma was diluted 1600 times in dilution buffer, and after the blocking reaction, the plate was washed three times with PBS, and then 50μl of diluted plasma sample was added to each well, followed by reaction (37°C, 3 hours). Upon completion of the reaction, the plate was washed three times with PBS, and 50 μl of secondary antibody, human total Ig-HRP (Southernbiotech, Cat. No. 1010-05), diluted 1000 times in dilution buffer, was added to each well, followed by reaction (37° C., 2 hours). The secondary antibody was then removed, the plate was washed five times with PBS, and 50 μl of color development reagent (TMB peroxidase substrate buffer, ROCKLAND, Cat. No. TMBE-1000) was added to each well, followed by color development for 15 minutes. Color development was stopped by adding 50 μl of 0.25 N HCl to each well, and the samples were analyzed at a wavelength of 450 nm using a microreader.

[0136] For the pseudovirus neutralization assay, the HEK293T-hACE2 (human angiotensin-converting enzyme 2; hACE2) cell line was cultured at 1x10 4 Plasma collected from monkeys by intravenous collection 2 and 3 weeks after immunization was diluted 4-fold from 100 to 6400 and incubated at 7x10 for 1 h (37°C). 5The plasma was reacted with TU / ml of pseudovirus (lentivirus expressing spike protein and luciferase). The plasma was inoculated into a 96-well plate containing HEK293T-hACE2 cells cultured with 2 μg / well (200 μl) of polybrene (Merck, Cat. No. TR-1003-G) and cultured for 2 days. The presence of neutralizing antibodies in the plasma that bind to the spike protein was then confirmed by measuring the degree of luciferase protein expression induced by infection of the HEK293T-hACE2 cell line with the pseudovirus. The cells cultured for 2 days were washed with DPBS and then lysed with 25 μg of cell lysis reagent (Promega, Cat. No. E153A). After transferring the lysed samples to an opaque 96-well plate, 100 μg of luciferase detection indicator (Promega, Cat. No. E151A) was added to each well and the level of luminescence was measured (injection of detection indicator-5 sec mixing-2 sec delay-10 sec measurement) using a luminometer (Luminometer Centro XS3 LB960, Berthold Technologies, Cat. No. LB960, software: Mikro 2000 program).

[0137] As a result, as shown in Figure 10, when the vector was injected, the amount of antibody production was excellent, and the antibody titer was confirmed to be maintained even 5 weeks after the vector injection. Furthermore, as shown in Figure 11, it was confirmed that the neutralizing antibody concentration was high when the vector #3 was injected. These results suggest that a large amount of neutralizing antibodies began to be produced 2 weeks after vaccination, efficiently blocking viral infection of cells.

[0138] <6-2> Comparison of hepatotoxicity in monkeys induced by injection of antigen expression vectors Due to the nature of adenovirus, many of them can migrate to the liver and cause hepatotoxicity.Hepatotoxicity is fatal to the human body, so it is very important to prepare a vaccine that does not show hepatotoxicity even when a high dose of viral particles is administered.Therefore, the hepatotoxicity is compared in monkeys by injection of the adenovirus vectors (vectors #1 and #3) prepared in Example 3.

[0139] Adenoviral vectors #1 and #3 prepared in Example 3 were administered to two monkeys per group at a dose of 1x10 11 A dose of VP / monkey was injected intramuscularly into 6-7 week-old monkeys (Macaca fascicularis). Blood was collected from all animals before dosing (pre) and 9, 22, and 36 days after dosing. All blood collections were performed in a fasting state (with free access to drinking water), and approximately 3 ml of blood was collected from the femoral vein using a disposable syringe (3 ml, 23 G, KOREAVACCINE, KOR).

[0140] Approximately 0.6 ml of blood collected for blood biochemistry was dispensed into SST™ tubes (Vacutainer®, BD, USA), and serum was separated by centrifugation (5424R, Eppendorf, USA) at 13,000 rpm for 5 min. Hepatotoxicity levels, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were then measured using a biochemistry analyzer (7180, HITACHI, JPN).

[0141] As a result, as shown in FIG. 12, it was confirmed that monkeys injected with vectors #1 and #3 using the same adenoviral vector platform did not show severe liver toxicity even when administered high doses of viral particles. EXAMPLES

[0142] Measurement of T cell reactivity when antigen-expressing vectors are injected into mice and monkeys <7-1> Measurement of T cell reactivity in spleen cells and peripheral blood mononuclear cells (PBMC) in mice T cell reactivity was measured in mice following injection of spike antigen-expressing vectors (Vector #1 and #3) prepared in Example 3 as follows.

[0143] Specifically, mice administered the vaccines of Example 5 (vectors #1 and #3) were sacrificed at week 10 of administration, blood was collected by retro-orbital blood collection, and spleens were isolated. Spleen cells were crushed to form single cells using a 70 μm cell strainer (BD Bioscience, Catalog No. 352350), and then red blood cells were removed using ACK lysis buffer (Gibco, Catalog No. A1049201). Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples by density gradient centrifugation using Histopaque®-1077 (Sigma-Aldrich, Catalog No. 10771). The isolated spleen cells and PBMCs were counted and 2×10 6 The cells were suspended in RF10 culture medium (RPMI1640 + 10% FBS + 1% penicillin / streptomycin) at a concentration of 100 cells / ml.

[0144] Peptivator SARS-CoV-2 Prot_S (Miltenyi Biotec, Cat. No. 130-126-701) was added to the pre-warmed RF10 culture medium at a concentration of 50 μg / ml, and 100 μl of the mixture was filled into each well of the coated plate in the Mouse IFN-γ ELISPOT Kit (CTL, Cat. No. MIFNgp-2M / 10) and stored in a CO2 incubator at 37°C for 20 minutes (each group of wells was performed in duplicate). Then, 100 μl of culture medium containing resuspended mouse spleen cells and PBMCs was added to each well and cultured in a CO2 incubator at 37°C. Anti-CD3 mAb (1 μg / ml, Biolegend, Cat. No. 100331) was added to the positive control wells, and only RF10 without cells was added to the negative control wells. After 24 hours of culture, ELISPOT development was performed according to the manufacturer's protocol included in the Mouse IFN-γ ELISPOT kit. After development, the plates were dried at room temperature for 24 hours by blocking light and then analyzed using an Immunospot S6 MicroAnalyzer (CTL). The results were analyzed using a 1x10 6 The total volume was converted to PBMC units.

[0145] As a result, as shown in Figure 13, it was confirmed that T cell reactivity was significantly increased in spleen cells and peripheral blood mononuclear cells (PBMCs) of mice injected with Vector #3 compared to the group not injected with the vector and the group injected with Vector #1. The above results suggest that the vaccine composition of the present invention can actively induce memory immune responses and antibody production by T cells when reinfected with SARS-CoV-2.

[0146] <7-2> Measurement of T cell reactivity in peripheral blood mononuclear cells (PBMC) in monkeys T cell reactivity was measured in monkeys following injection of the adenoviral vectors (vectors #1 and #3) prepared in Example 3 as follows.

[0147] Blood (5 ml) was collected from monkeys administered the vaccine of Example 6 (Vector #3) by intravenous blood sampling on week 4 of dosing. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples by density gradient centrifugation using Histopaque®-1077 (Sigma-Aldrich, Catalog No. 10771) followed by removal of red blood cells using ACK lysis buffer (Gibco, Catalog No. A1049201). Isolated PBMCs were counted and 2x10 6 The cells were suspended in RF10 culture medium (RPMI1640 + 10% FBS + 1% penicillin / streptomycin) at a concentration of 100 cells / ml.

[0148] Peptivator SARS-CoV-2 Prot_S (Miltenyi Biotec, Cat. No. 130-126-701) was added to pre-warmed RF10 culture medium at a concentration of 100 μg / ml, and 100 μl of the mixture was filled into each well of the coated plate in the Human IFN-γ ELISPOT Kit (CTL, Cat. No. HIFNgp-2M / 10) and stored in a CO2 incubator at 37°C for 20 minutes (each group of wells was performed in duplicate). Then, 100 μl of culture medium containing resuspended PBMCs was added to each well and cultured in a CO2 incubator at 37°C. PMA (10 ng / ml) and ionomycin (1 μg / ml) were added to the positive control wells, and only RF10 was added to the negative control wells without cells. After 24 hours of culture, ELISPOT development was performed according to the manufacturer's protocol included in the Human IFN-γ ELISPOT kit. After development, the plates were dried at room temperature for 24 hours by blocking light and then analyzed using an Immunospot S6 MicroAnalyzer (CTL). The results were analyzed using a 1x10 6 The total volume was converted to PBMC units.

[0149] As a result, as shown in Figure 14, it was confirmed that T cell reactivity was significantly increased in peripheral blood mononuclear cells (PBMCs) of monkeys injected with Vector #3 compared to the group not injected with the vector. The above results suggest that the vaccine composition of the present invention can actively induce memory immune responses and antibody production by T cells in the case of reinfection with SARS-CoV-2. EXAMPLES

[0150] Comparison of the equivalence of antigen-specific neutralizing antibody production in mice and monkeys by injection of antigen expression vector #3 and antigen hetero vector #3 <8-1> Comparison of the equivalence of antigen-specific neutralizing antibody production in mice by injection of antigen expression vector #3 and antigen hetero vector #3 The amount of antibody production after injecting mice with the vector #3 prepared in Example 3 and the hetero vector #3 (SEQ ID NO: 30) prepared as an equivalent comparison group was compared. The hetero vector #3 is a form in which the E4 gene in the adenovirus gene of the vector #3 used in Example 3 is rearranged into the E1 region of the viral genome. Specifically, this vector is a vector in which the E3 knob gene is replaced with Ad35 in E1 / E3 deleted Ad5, 711 bp of E4orf6 of the Ad5 / 35 vector is deleted, and the recombinant spike protein (the S1 and S2 domains of SARS-CoV-2 are linked with the GGGGS linker) is inserted into the Ad5 / 35 vector. (SEQ ID NO:31) The vector was prepared in the same manner as in Example 3.

[0151] Specifically, spike antigen expression vectors (vector #3 and heterologous vector #3) were administered at a dose of 1x10 9VP / mouse was intramuscularly injected into 6-7 week old BALB / c mice. Approximately 300 μl of blood was collected from the mice by retro-orbital blood collection 9 weeks after administration. Plasma was then separated by centrifugation (8000 rpm, 10 min, 20°C) and the amount of neutralizing antibodies present in the blood was measured by pseudovirus neutralization assay.

[0152] For the pseudovirus neutralization assay, the HEK293T-hACE2 (human angiotensin-converting enzyme 2; hACE2) cell line was cultured at 1x10 4 Cells / well were plated in 96-well plates at a density of 100 cells / well and incubated for 10 hours (37°C, 5% CO2). Plasma collected from mice by intravenous collection 8 weeks after immunization was diluted 4-fold from 100 to 6400, and 7x10 5 The cells were reacted with TU / ml of pseudovirus (lentivirus expressing spike protein and luciferase) for 1 hour (37°C) and then cultured in 96-well plates containing HEK293T-hACE2 cells for 3 days. The presence of neutralizing antibodies in the plasma that bind to the spike protein was then confirmed by measuring the degree of luciferase protein expression induced by infection of the HEK293T-hACE2 cell line with the pseudovirus. The cells cultured for 3 days were washed with DPBS and then lysed with 25 μg of cell lysis reagent (Promega, catalog number E153A). After transferring the lysed samples to an opaque 96-well plate, 100 μg of luciferase detection indicator (Promega, Cat. No. E151A) was added to each well and the level of luminescence was measured (injection of detection indicator-5 sec mixing-2 sec delay-10 sec measurement) using a luminometer (Luminometer Centro XS3 LB960, Berthold Technologies, Cat. No. LB960, software: Mikro 2000 program).

[0153] As a result, as shown in Figure 15, antigen vector #3 and antigen hetero vector #3 showed excellent antibody production, and there was no difference between the two groups. The above results suggest that antigen #3 has a similar level of neutralizing antibody activity even when loaded onto a different platform vector.

[0154] <8-2> Comparison of the equivalence of antigen-specific neutralizing antibody production in monkeys by injection of antigen expression vector #3 and antigen hetero vector #3 Vector #3 and heterologous vector #3 prepared as an equivalent comparison group in Example 8-1 were injected into monkeys (cynomolgus monkeys), and the amounts of antigen-specific neutralizing antibodies produced were compared.

[0155] Specifically, spike antigen expression vectors (vector #3 and heterologous vector #3) were administered at 2x10 10 A dose of VP / monkey was injected intramuscularly into 6-7 week old monkeys. Blood was collected from the monkeys by intravenous blood sampling 9 weeks after immunization. Plasma was then separated by centrifugation (8000 rpm, 10 min, 20°C) and the amount of neutralizing antibodies present in the blood was measured by pseudovirus neutralization assay.

[0156] For the pseudovirus neutralization assay, the HEK293T-hACE2 (human angiotensin-converting enzyme 2; hACE2) cell line was cultured at 1x10 4 The cells were plated in 96-well plates at a density of 100 cells / well and incubated for 10 hours (37°C, 5% CO2). Plasma collected from monkeys by intravenous blood sampling 9 weeks after immunization was diluted 4-fold from 100 to 6400, and 7x10 5The cells were reacted with TU / ml of pseudovirus (lentivirus expressing spike protein and luciferase) for 1 hour (37°C) and then cultured in 96-well plates containing HEK293T-hACE2 cells for 3 days. The presence of neutralizing antibodies in the plasma that bind to the spike protein was then confirmed by measuring the degree of luciferase protein expression induced by infection of the HEK293T-hACE2 cell line with the pseudovirus. The cells cultured for 3 days were washed with DPBS and then lysed with 25 μg of cell lysis reagent (Promega, catalog number E153A). After transferring the lysed samples to an opaque 96-well plate, 100 μg of luciferase detection indicator (Promega, Cat. No. E151A) was added to each well and the level of luminescence was measured (injection of detection indicator-5 sec mixing-2 sec delay-10 sec measurement) using a luminometer (Luminometer Centro XS3 LB960, Berthold Technologies, Cat. No. LB960, software: Mikro 2000 program).

[0157] As a result, as shown in FIG. 16, vector #3 and heterologous vector #3 showed excellent antibody production, with no difference in expression levels. EXAMPLES

[0158] Comparison of antibody production in mice by recombinant spike protein injection <9-1> Comparison of mouse antibody production by injection of recombinant spike protein The recombinant spike proteins (recombinant protein #2 and recombinant protein #3) derived from vectors #1 to #3 prepared in Example 3 were injected into mice, and the amounts of antibody production were compared.

[0159] Specifically, recombinant spike proteins derived from vectors #1-#3 were injected intramuscularly into 6-7 week-old BALB / c mice, with 6 mice per group. 10 μg of each recombinant spike protein (#1-#3) was diluted in 150 μl of PBS (pH 7.4) and administered by intramuscular injection. Approximately 300 μl of blood samples were collected by retro-orbital blood collection 2-4 weeks after administration. Plasma was then separated by centrifugation (8000 rpm, 10 min, 20°C), and the amount of neutralizing antibodies present in the blood was measured by enzyme-linked immunosorbent assay (ELISA) and pseudovirus neutralization assay.

[0160] For enzyme-linked immunosorbent assay, 96-well plates were coated with spike protein (Acro Biosystems, Cat. No. SPN-C52H84) dissolved in PBSN (PBS 1L + sodium azide 0.01g) at 100ng / well, followed by cold reaction at 4°C for 16 hours. After coating the 96-well plates for 16 hours, the coating protein was removed, washed three times with PBS, and then 150μl of blocking buffer (PBSN+BSA 1%) was added to each well, followed by reaction (37°C, 90 minutes). During the reaction time, plasma was diluted 6400 times in dilution buffer (PBSN+0.1%BSA+0.05%Tween-20), and after the blocking reaction, the plate was washed three times with PBS, and then 50μl of diluted plasma sample was added to each well, followed by reaction (37°C, 3 hours). Upon completion of the reaction, the plate was washed three times with PBS, and 50 μl of secondary antibodies, GAM-IgG-HRP (Southernbiotech, Cat. No. 1030-05) and GAM-IgM-HRP (Southernbiotech, Cat. No. 1020-05), diluted 1000 times in dilution buffer, were added to each well, followed by reaction (37° C., 2 hours). The secondary antibody was then removed, the plate was washed five times with PBS, and 50 μl of color development reagent (TMB peroxidase substrate buffer, ROCKLAND, Cat. No. TMBE-1000) was added to each well, followed by color development for 15 minutes. Color development was stopped by adding 50 μl of 0.25 N HCl to each well, and the samples were analyzed at a wavelength of 450 nm using a microreader.

[0161] For pseudovirus neutralization assays, HEK293T-hACE2 (human angiotensin-converting enzyme 2; hACE2) cell lines were cultured at 1x10 4 Plasma collected from mice by intravenous collection 2-4 weeks after immunization was diluted 4-fold from 1:100 to 1:6400 and diluted 7x10 5The cells were reacted with TU / ml of pseudovirus (lentivirus expressing spike protein and luciferase) for 1 hour (37°C) and then cultured in 96-well plates containing HEK293T-hACE2 cells for 3 days. The presence of neutralizing antibodies in the plasma that bind to the spike protein was then confirmed by measuring the degree of luciferase protein expression induced by infection of the HEK293T-hACE2 cell line with the pseudovirus. The cells cultured for 3 days were washed with DPBS and then lysed with 25 μg of cell lysis reagent (Promega, catalog number E153A). After transferring the lysed samples to an opaque 96-well plate, 100 μg of luciferase detection indicator (Promega, Cat. No. E151A) was added to each well and the level of luminescence was measured (injection of detection indicator-5 sec mixing-2 sec delay-10 sec measurement) using a luminometer (Luminometer Centro XS3 LB960, Berthold Technologies, Cat. No. LB960, software: Mikro 2000 program). EXAMPLES

[0162] Comparison of antibody production in mice by spike antigen plasmid injection <10-1> Comparison of mouse antibody production by spike antigen plasmid injection Vectors #1 to #3 prepared in Examples 1 and 2 in the form of plasmids (plasmid vector #1 and plasmid vector #3) were injected into mice, and the amount of antibody production was compared.

[0163] Specifically, the plasmid vectors #2 and #3 prepared in Examples 1 and 2 were introduced into E. coli and cultured in 200 mL of LB culture medium for one day. The cultured plasmids were harvested using a QIAGEN Endofree Plasmid Maxi kit (catalog number 12362) and then administered to 6-7 week-old BALB / c mice with 6 mice per group. 100 μg of each vector (#2 and #3) was diluted in 50 μl of PBS (pH 7.4) and administered by intramuscular injection. Approximately 300 μl of blood samples were collected by orbital blood collection 2-4 weeks after administration. Plasma was then separated by centrifugation (8000 rpm, 10 min, 20°C), and the amount of neutralizing antibodies present in the blood was measured by enzyme-linked immunosorbent assay (ELISA) and pseudovirus neutralization assay.

[0164] For enzyme-linked immunosorbent assay, 96-well plates were coated with spike protein (Acro Biosystems, Cat. No. SPN-C52H84) dissolved in PBSN (PBS 1L + sodium azide 0.01g) at 100ng / well, followed by cold reaction at 4°C for 16 hours. After coating the 96-well plates for 16 hours, the coating protein was removed, washed three times with PBS, and then 150μl of blocking buffer (PBSN+BSA 1%) was added to each well, followed by reaction (37°C, 90 minutes). During the reaction time, plasma was diluted 6400 times in dilution buffer (PBSN+0.1%BSA+0.05%Tween-20), and after the blocking reaction, the plate was washed three times with PBS, and then 50μl of diluted plasma sample was added to each well, followed by reaction (37°C, 3 hours). Upon completion of the reaction, the plate was washed three times with PBS, and 50 μl of secondary antibodies, GAM-IgG-HRP (Southernbiotech, Cat. No. 1030-05) and GAM-IgM-HRP (Southernbiotech, Cat. No. 1020-05), diluted 1000 times in dilution buffer, were added to each well, followed by reaction (37° C., 2 hours). The secondary antibody was then removed, the plate was washed five times with PBS, and 50 μl of color development reagent (TMB peroxidase substrate buffer, ROCKLAND, Cat. No. TMBE-1000) was added to each well, followed by color development for 15 minutes. Color development was stopped by adding 50 μl of 0.25 N HCl to each well, and the samples were analyzed at a wavelength of 450 nm using a microreader.

[0165] For pseudovirus neutralization assays, HEK293T-hACE2 (human angiotensin-converting enzyme 2; hACE2) cell lines were placed in 96-well plates at a density of 1x104 cells / well and cultured for 10 h (37°C, 5% CO2). Plasma collected from mice by intravenous collection 2-4 weeks after immunization was diluted 4-fold from 100-1000 to 6400-1000, reacted with 7x105 TU / ml of pseudovirus (lentivirus expressing spike protein and luciferase) for 1 h (37°C), and then cultured for 3 days in 96-well plates containing HEK293T-hACE2 cells. The presence of neutralizing antibodies in the plasma that bind to the spike protein was then confirmed by measuring the degree of luciferase protein expression induced by infection of the HEK293T-hACE2 cell line with the pseudovirus. The cells cultured for 3 days were washed with DPBS and then lysed with 25 μg of cell lysis reagent (Promega, Cat. No. E153A). After transferring the lysed samples to an opaque 96-well plate, 100 μg of luciferase detection indicator (Promega, Cat. No. E151A) was added to each well and the level of luminescence was measured using a luminometer (Luminometer Centro XS3 LB960, Berthold Technologies, Cat. No. LB960, software: Mikro 2000 program) (injection of detection indicator-mixing 5 sec-delay 2 sec-measurement 10 sec). The present disclosure includes the following embodiments. [1] A recombinant spike protein in which the cleavage site between the S1 and S2 genes of the coronavirus spike protein has been removed and a linker sequence has been introduced. [2] The recombinant spike protein of embodiment 1, wherein the coronavirus is SARS-CoV, MERS-CoV or SARS-CoV-2. [3] The recombinant spike protein of embodiment 2, wherein the coronavirus is SARS-CoV-2. [4] The recombinant spike protein of embodiment 1, wherein the linker is composed of (GGGGS)n. [5] The recombinant spike protein of embodiment 4, wherein n is an integer from 1 to 5. [6] A recombinant spike protein described in any one of embodiments 1 to 5, wherein the spike protein antigen linked by the linker sequence has increased stability. [7] A recombinant spike protein according to any one of embodiments 1 to 5, wherein the spike protein antigen linked by the linker sequence has an increased antigen expression level. [8] A polynucleotide encoding a recombinant spike protein in which the cleavage site between the S1 and S2 genes of the coronavirus spike protein has been removed and a linker sequence has been introduced. [9] The polynucleotide of embodiment 8, wherein the coronavirus is SARS-CoV, MERS-CoV or SARS-CoV-2.

[10] The polynucleotide of embodiment 8, wherein the linker sequence is composed of (GGGGS)n.

[11] A vector containing a polynucleotide encoding a recombinant spike protein in which the cleavage site between the S1 and S2 genes of the coronavirus spike protein has been removed and a linker sequence has been introduced.

[12] The vector of embodiment 11, wherein the coronavirus is SARS-CoV, MERS-CoV or SARS-CoV-2.

[13] The vector of embodiment 12, wherein the coronavirus is SARS-CoV-2.

[14] The vector of embodiment 11, which is a virus or a plasmid.

[15] The vector of embodiment 14, wherein the virus is an adenovirus, a retrovirus, a lentivirus, a modified vaccinia virus Ankara (MVA), an adeno-associated virus (AAV), a herpes simplex virus or a baculovirus.

[16] The vector of embodiment 15, wherein the adenovirus is Ad2, Ad4, Ad5, Ad11, Ad26, Ad35, ChAd68, FAd9 or PAd3.

[17] The vector of embodiment 15 or 16, wherein the adenovirus has a high antigen expression level.

[18] A vaccine for preventing or treating coronavirus infection, comprising a vector containing polynucleotides of the S1 and S2 genes of the coronavirus spike protein.

[19] The vaccine for preventing or treating a coronavirus infection described in embodiment 18, wherein the coronavirus infection is SARS, MERS or COVID-19.

[20] A method for preventing or treating coronavirus infection, comprising administering to a subject a vector containing polynucleotides of the S1 and S2 genes of the coronavirus spike protein.

Claims

1. A vector for expressing a recombinant spike protein from a coronavirus, the vector comprising a polynucleotide encoding a recombinant spike protein in which the amino acid sequence TNSPRRAR of the cleavage site between the S1 and S2 genes of the spike protein is replaced by a linker sequence having the amino acid sequence TGGGGSR; The vector is derived from an adenoviral vector comprising the sequence set forth in SEQ ID NO: 21, the E1 and E3 regions of the adenoviral vector are deleted, and The vector, wherein a polynucleotide encoding the recombinant spike protein is inserted into the deleted E1 region.

2. The vector of claim 1 , wherein the coronavirus is SARS-CoV, MERS-CoV or SARS-CoV-2.

3. The vector of claim 2, wherein the coronavirus is SARS-CoV-2.

4. The vector of claim 1 , wherein the polynucleotide has the polynucleotide sequence set forth in SEQ ID NO:

12.

5. 2. The vector of claim 1 , wherein the vector expressing the recombinant spike protein has a polynucleotide sequence as set forth in SEQ ID NO:

27.

6. A vaccine for preventing or treating a coronavirus infection comprising the vector of claim 1.

7. The vaccine for preventing or treating a coronavirus infection according to claim 6, wherein the coronavirus infection is SARS, MERS or COVID-19.

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