Vaccine preparation to prevent SARS-CoV-2

Abstract

The invention relates to a vaccine preparation for the prevention of SARS-CoV-2, including the mRNA encoding the SARS-CoV-2 S mutant antigen, in which the SARS-CoV-2 vaccine according to the invention exhibits high stability and immunogenicity in the body, is easy to store and use, and can be expected to have excellent preventive effect against COVID-19.

Description

SARS-COV-2 preventive vaccine composition

[0001] The present invention relates to a SARS-CoV-2 preventive vaccine composition, and more particularly, to a SARS-CoV-2 preventive vaccine composition comprising mRNA encoding an S variant antigen of the SARS-CoV-2 virus.

[0002]

[0003] Coronaviruses are a type of RNA virus, whose genetic information consists of ribonucleic acid (RNA). They cause respiratory and digestive infections in humans and animals. They are easily transmitted through mucosal contact and droplet transmission. In humans, they typically cause mild respiratory infections, but in rare cases, they can cause fatal infections.

[0004] The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is currently causing a pandemic, is a positive-sense single-stranded RNA coronavirus based on its genetic sequence. It is infectious to humans and is the cause of coronavirus disease 2019 (COVID-19). The SARS-CoV-2 virus uses the spike protein on its surface to bind to angiotensin-converting enzyme 2 (ACE2) present on the surface of airway epithelial cells, alveolar epithelial cells, vascular endothelial cells, and macrophages in the lungs, and invades host cells. A study on the genome sequence of SARS-CoV-2 identified a receptor-binding domain (RBD) within the spike protein that has a tertiary structure quite similar to that of SARS-CoV, and it was speculated that the RBD of SARS-CoV-2 has a higher binding affinity to ACE2 than that of SARS-CoV, suggesting that SARS-CoV-2 is more infectious than SARS-CoV (Matthew Z Tet al., Natreal reviews immunology, 20:363-374, 2020).

[0005] The spike protein is composed of two proteins, S1 and S2, of which the S1 protein consists of an amino-terminal domain and an RBD. When the RBD binds to ACE2, the SARS-CoV-2 virion enters the endosome of the cell through endocytosis, after which the fusion peptide is exposed and inserted into the membrane of the host cell. The S2 protein consists of a fusion peptide region (FP region) and heptad repeat regions (HR1, HR2), and the HR1 and HR2 fuse with the viral membrane in a form where they touch each other, and the SARS-CoV-2 virion is released out of the host cell. S1 and S2 have different cleavage sites and are cleaved by their respective proteases, which causes SARS-CoV-2 infection. Therefore, treatments and vaccines for SARS-CoV-2 are being developed using strategies that inhibit S1 and S2 cleavage or interfere with the binding between the virus and proteins called ACE2 or TMPRSS2 (transmembrane serine protease 2) (Mattew Z Tet al., Naturopathic Reviews Immunology, 20:363-374, 2020).

[0006] The spike protein of SARS-CoV-2 has an unstable protein structure, so proline substitutions at positions 986 (K) and 987 (V) prevent misfolding or triggering, and thus the prefusion stabilized viral glycoprotein acts as a superior immunogen. This has been confirmed through previous studies on MERS-CoV and SARS-CoV (Jesper Pallesenet et al. PNAS, 2017, DOI: https: / / doi.org / 10.1073 / PNAS.1707304114).

[0007] Meanwhile, SARS-CoV-2 has mutated from the 'D type (D614)' that originated in Wuhan to the 'European type' or 'G type (G614)' according to the global initiative on sharing avian influenza data (GISAID). The G type mutation is characterized by a mutation of amino acid 614 of the viral surface spike protein from aspartic acid (GAT; Asp, D) to glycine (GGT; Gly, G) (Fig. 1) (Plante, J.Aet al. Nature (2020). DOI: https: / / doi.org / 10.1038 / s41586-020-2895-3).

[0008] Amid the global pandemic, vaccines to prevent SARS-CoV-2 virus infection are being developed and administered, but there is still a need for vaccines with more stable and sustained effects and high immunogenicity.

[0009] Meanwhile, gene therapy and genetic vaccines are already proven and generally applicable technologies in the medical field, and can be used to treat not only genetic diseases but also autoimmune diseases, infectious diseases, cancer or tumor-related diseases, and inflammatory diseases.

[0010] Genetic vaccines began to be developed when it was reported that when DNA and RNA encoding target genes were directly injected into animals, the target genes were expressed in living animals, and immunity was possible through this expression (Wolff JA et al. Science, 247:1465-8, 1990).

[0011] Genetic vaccination allows for the induction of a desired immune response against selected antigens, such as characteristic components of bacterial surfaces, viral particles, and tumor antigens. Broadly speaking, vaccination is one of the pivotal achievements of modern medicine. However, effective vaccines are currently available for only a limited number of diseases. Consequently, infections that cannot be prevented by vaccination still affect millions of people each year.

[0012] In gene therapy or genetic vaccination, DNA can be used as a nucleic acid molecule for gene administration, and DNA is known to be relatively stable and easier to handle compared to RNA. However, in the case of DNA, potential risks arise if the administered DNA fragment inserts into an unintended location within the patient's genome, causing genetic damage. Additionally, unwanted anti-DNA antibodies may develop. Another problem is the limited expression level of peptides or proteins produced by DNA administration and subsequent transcription / translation. The presence or absence of specific transcription factors that regulate DNA transcription significantly influences the expression level of administered DNA. In the absence of specific transcription factors, sufficient RNA is not produced by DNA transcription, resulting in limited levels of peptides or proteins produced through translation.

[0013] On the other hand, when RNA is used as a tool for gene delivery, RNA does not require transcription and can synthesize proteins directly within the cytoplasm without the need for entry into the nucleus like DNA, eliminating the risk of RNA incorporation into cellular chromosomes and causing unwanted genetic damage. Furthermore, its short half-life compared to DNA means it does not induce long-term genetic alterations (Sayour EJ, et al., J Immunother Cancer 2015;3:13, 2015). When delivered into cells, typical RNA vaccines are activated for a short period of time to express the target protein, and are destroyed within a few days by enzymatic reactions, while a specific immune response to the expressed target antigen (protein) remains.

[0014] Furthermore, when using RNA as a gene delivery tool, it works by passing only through the cell membrane, without the need to cross the nuclear membrane. Therefore, even with smaller amounts, the same amount of target protein can be expressed. Furthermore, RNA possesses inherent immunogenic properties, allowing for the same immune effect to be achieved with smaller doses compared to DNA.

[0015] By using RNA instead of DNA for genetic vaccination, the risk of unwanted genome integration and the generation of anti-DNA antibodies can be minimized or prevented. However, RNA is a highly unstable molecular species that can be easily degraded by ubiquitous RNases.

[0016] While significant advances have been made over the past several years, there remains a need for an efficient method for delivering mRNA vaccines capable of eliciting an immune response, without compromising efficacy due to inefficient translation of the mRNA resulting from premature antigen degradation or inefficient mRNA release from cells. Furthermore, there is a need to reduce the dose of mRNA vaccines to mitigate potential safety concerns and make them affordable in developing countries.

[0017] Many challenges remain when it comes to delivering nucleic acids to achieve desired responses in biological systems. Nucleic acid-based therapeutics hold tremendous promise, but realizing this potential requires effective delivery of nucleic acids to the appropriate site within a cell or organism.

[0018] However, the use of nucleic acids for therapeutic and prophylactic purposes currently faces two challenges. First, free RNA is vulnerable to nuclease degradation in plasma. Second, free RNA has limited access to intracellular compartments where the translational machinery resides. Attempts are being made to introduce lipid nanoparticles formed from cationic lipids, along with other lipid components such as neutral lipids, cholesterol, PEG, PEGylated lipids, and oligonucleotides, to block RNA degradation in plasma and promote cellular uptake of nucleic acids.

[0019] Accordingly, the inventors of the present invention have made great efforts to develop a preventive vaccine against SARS-CoV-2 with excellent storage stability and immunogenicity in the body, and as a result, they have developed an mRNA vaccine in which a nucleic acid encoding a mutant antigen for the spike protein of SARS-CoV-2 is loaded onto a lipid nanoparticle (LNP: Lipid nanoparticle) or liposome having a specific lipid composition, and confirmed that the vaccine exhibits excellent stability and high immunogenicity in vivo, thereby completing the present invention.

[0020]

[0021] Summary of the invention

[0022] The purpose of the present invention is to provide a SARS-CoV-2 preventive vaccine composition exhibiting excellent stability and high immunogenicity, named EG-COVID.

[0023] To achieve the above purpose, the present invention provides a vaccine composition for preventing SARS-CoV-2 comprising mRNA encoding the S antigen of the SARS-CoV-2 virus.

[0024] The vaccine composition according to the present invention may additionally include liposomes or lipid nanoparticles, and the liposomes or lipid nanoparticles may include cationic lipids, neutral lipids, and cholesterol.

[0025] The present invention also provides a method for preventing SARS-CoV-2 infection, comprising administering a composition for preventing SARS-CoV-2 comprising mRNA encoding the S antigen of the SARS-CoV-2 virus.

[0026] The present invention also provides the use of a composition comprising mRNA encoding the S antigen of the SARS-CoV-2 virus for the prevention of SARS-CoV-2 infection.

[0027] The present invention also provides the use of a composition for preventing SARS-CoV-2 infection comprising mRNA encoding the S antigen of the SARS-CoV-2 virus for the manufacture of a medicament for preventing SARS-CoV-2 infection.

[0028]

[0029] Figure 1 shows the D (D614) and G (G614) mutations of SARS-CoV-2.

[0030] Figure 2 shows the results of confirming the delivery efficiency of liposomes with different lipid compositions into mouse materials.

[0031] Figure 3 shows the mRNA expression efficiency in mice according to the mixing ratio of mRNA and liposome in the mRNA-ribosome complex.

[0032] Figure 4 shows the results of confirming whether the formulation mixing CV-SF-614Gm with CV-LP-b1 is normally expressed in HEK293T cells. Figure 4A shows the results of confirming SARS-CoV-2 Spike protein expression after treating HEK293 cells with CV-SF-614Gm at various concentrations using lipofectamine, and Figure 4B shows the results of confirming SARS-CoV-2 Spike protein expression after treating HEK293T cells with an mRNA complex prepared by mixing CV-LP-b1 and CV-SF-614Gm and lyophilizing it.

[0033] Figure 5A shows the antibody titer of RBD-specific IgG analyzed by ELISA in the serum of mice administered the mRNA complex of CV-SF-614Gm mixed with CV-LP-b1, Figure 5B shows the concentration of IFN-γ secreted in the medium after stimulating splenocytes isolated from mice administered the mRNA complex of CV-SF-614Gm mixed with CV-LP-b1 with S1 peptide, and Figure 5C shows the neutralizing antibody formation ability analyzed using the SARS-CoV-2 surrogate virus neutralization test (sVNT) Kit of the obtained serum.

[0034] Figure 6A shows the antibody titer of RBD-specific IgG analyzed by ELISA in the serum of mice that were administered a single or two doses of the mRNA complex of CV-SF-614Gm mixed with CV-LP-b1, Figure 6B shows the concentration of IFN-γ secreted into the medium after stimulating splenocytes isolated from mice that were administered a single or two doses of the mRNA complex of CV-SF-614Gm mixed with CV-LP-b1 with S1 peptide, and Figure 6C shows the neutralizing antibody formation ability analyzed using the SARS-CoV-2 surrogate virus neutralization test (sVNT) Kit of the obtained serum.

[0035] Figure 7A shows the antibody titer of RBD-specific IgG analyzed by ELISA in the sera of mice administered with L-EG-COVID, a liquid formulation, and F-EG-COVID, a lyophilized formulation, after refrigeration storage for 0, 4, and 8 weeks, respectively. Figure 7B shows the concentration of IFN-γ secreted in the medium after stimulating the splenocytes of mice immunized by the above method with S1 peptide, analyzed by ELISA, and Figure 7C shows the neutralizing antibody formation ability analyzed using the SARS-CoV-2 surrogate virus neutralization test (sVNT) Kit of the obtained sera.

[0036] Figures 8a and 8b show the results of examining the body distribution pattern of CV-SF-614Gm over time after intramuscular administration of EG-COVID to rats.

[0037] Figure 9 shows the results of evaluating the neutralizing antibody titer of EG-COVID. A shows the results of deriving the IC50 value of EG-COVID based on the infection inhibition rate of the SARS-CoV-2 (NCCP 43326) virus according to the concentration of CV-SF-614Gm mRNA, and B shows the IC 50 value of EG-COVID against the alpha mutant virus and the beta mutant virus.

[0038]

[0039] Detailed description of the invention and preferred embodiments

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0041]

[0042] The present invention relates to a vaccine composition for preventing SARS-CoV-2 comprising mRNA encoding the S antigen of the SARS-CoV-2 virus.

[0043] In the present invention, the S antigen of the SARS-CoV-2 virus is used as a concept including a wild-type S antigen and a variant S antigen including one or more amino acid mutations.

[0044] In particular, the S antigen of the SARS-CoV-2 virus in the present invention is preferably a sequence (CV-SF-614Gm) in which the 614G variant of the spike protein is used as a backbone to stabilize the spike protein structure, and 986 (K) and 987 (V) are additionally substituted with proline and / or RRAR, which is an amino acid sequence of 682 to 685, is mutated to QQAQ, in order to stabilize the spike protein structure.

[0045] In addition, sequence optimization was performed to increase the content of guanine and cytosine in the mRNA encoding the S antigen of the SARS-CoV-2 virus to stabilize the mRNA and increase translation efficiency in humans. In particular, among the indicators that can predict RNA stability, RNA fold, RNA fold thermodynamic ensemble, RNA structure, and thermodynamic energy of cofold were confirmed, and based on the results, CV-SF-WT-614D (mRNA encoding SARS-CoV-2 spike protein, SEQ ID NO: 1) and CV-SF-614Gm (mRNA encoding the spike protein of SARS-CoV-2 614G variant, SEQ ID NO: 2) with the lowest △G value were selected (Table 1).

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056] The vaccine composition of the present invention may additionally contain a liposome or a lipid nanoparticle (LNP, lipid nanoparicle), and the mRNA encoding the S antigen of the SARS-CoV-2 virus may be adsorbed or associated to the outside of the liposome or lipid nanoparticle, or encapsulated or enclosed inside the liposome or lipid nanoparticle.

[0057] The liposome or lipid nanoparticle contained in the vaccine composition of the present invention comprises a cationic lipid, and may preferably additionally comprise a neutral lipid.

[0058] The cationic lipids include dimethyldioctadecylammonium bromide (DDA), C12-200, 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 3β-[N-(N′,N′-dimethylaminoethane) carbamoyl cholesterol (DC-Chol), 1,2-dioleoyloxy-3-dimethylammoniumpropane (DODAP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1 Etyle PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:1 Ethyl PC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1 Ethyl PC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholin (18:0 Ethyl PC), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (16:0 Ethyl PC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0 Ethyl PC), 1,2-dilauroyl-sn-glycero-3-ethylphosphocholin (12:0 Ethyl PC),N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dimyristoyl-3-dimethylammonium-propane (14:0 DAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (16:0 DAP), 1,2-distearoyl-3-dimethylammonium-propane (18:0 DAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 1,2-stearoyl-3-trimethylammonium-propane (18:0 TAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (16:0 TA), Preferably, at least one selected from the group consisting of 1,2-dimyristoyl-3-trimethylammonium-propane (14:0 TAP) and N4-cholesteryl-spermine (GL67), but is not limited thereto, most preferably, C12-200 or 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), but is not limited thereto.

[0059] Although cationic liposomes are generally known to be toxic, the vaccine composition according to the present invention has the characteristic of losing toxicity due to adsorption of mRNA (Filion, MC, & Phillips, NC, Biochimica et Biophysica Acta (BBA) - Biomembranes, 1329(2), 345-356. 1997).

[0060] The liposome or lipid nanoparticle comprising a cationic lipid according to the present invention may additionally comprise a neutral lipid.

[0061] The above neutral lipids are 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), phosphatidylserine (PS), phosphoethanolamine (PE), phosphatidylglycerol (PG), phosphoric acid (PA), phosphatidylcholine (PC), and 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol) (DOPI), 1,2-distearoyl-sn-glycero-3-phosphoinositol (DSPI), but is not limited thereto, and most preferably It may be, but is not limited to, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0062] The liposome or lipid nanoparticle according to the present invention may additionally include one or more delivery factors selected from the group consisting of protamine, albumin, transferrin, PTD (protein transduction domains), CPP (cell penetrating peptide), Polyethylene glycol (PEG), Pegylated lipid, metal ion-bound lipid, and Macrophage targeting moiety.

[0063] In the present invention, "DOTAP (Dioleoyl-3-trimethylammonium propane)", which is an example of a preferred cationic lipid, is a cationic emulsifier having a structure of chemical formula 1, and is used as a fabric softener. Recently, it has been used as a carrier for proteins, compounds, peptides, etc., as well as a nucleic acid carrier forming liposomes or lipid nanoparticles.

[0064]

[0065] [Chemical Formula 1]

[0066]

[0067] Also, in the present invention, "DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine)", which is an example of a preferred neutral lipid, has a structure of chemical formula 2 and is used as an auxiliary lipid for forming cationic liposomes or lipid nanoparticles.

[0068] [Chemical Formula 2]

[0069]

[0070] In the liposome or lipid nanoparticle according to the present invention, the weight ratio of the cationic lipid and the neutral lipid may be 1:9 to 9.5:0.5, preferably 2:8 to 9:1, more preferably 3:7 to 8:2, and most preferably 4:6 to 7:3.

[0071]

[0072] In the present invention, the liposome or lipid nanoparticle according to the present invention may additionally contain cholesterol. In the liposome or lipid nanoparticle of the present invention, the weight ratio of the cationic lipid to the cholesterol may be, but is not limited to, 6:1 to 1:3, preferably 4:1 to 1:2.5, more preferably 3:1 to 1:2, and most preferably 2.5:1 to 1:1.5.

[0073] In addition, in the present invention, when the liposome or lipid nanoparticle according to the present invention includes all of cationic lipid, neutral lipid, and cholesterol, the weight ratio of the cationic lipid, neutral lipid, and cholesterol may be 1 to 9.5:0.5 to 9:0.05 to 3, preferably 3 to 8:7 to 1:0.45 to 7.0, and more preferably 1 to 3.5:1 to 3.5:0.5 to 3, but is not limited thereto.

[0074]

[0075] In one embodiment of the present invention, the weight ratio of cationic lipid, neutral lipid, and cholesterol is 2:2:1 (40:40:20 w / w / w), but is not limited thereto.

[0076] When cholesterol is additionally included, for example, when DOTAP:DOPE is used at a weight ratio of 1:1, liposomes or lipid nanoparticles can be prepared by mixing cholesterol at a weight ratio of 0.2 to 0.85, preferably 0.4 to 0.6, relative to DOTAP.

[0077] Additionally, in the SARS-CoV-2 preventive vaccine composition according to the present invention, the mixing ratio of liposomes or lipid nanoparticles and mRNA can be expressed as the N:P ratio, and the mRNA expression and stability of the composition are affected depending on the N:P ratio.

[0078] In the present invention, the N:P ratio of the liposome or lipid nanoparticle and mRNA may be characterized as being 0.2:1 to 1.4:1, preferably 0.23:1 to 1.0:1, more preferably 0.46:1 to 1.0:1, and in the present invention, an N:P ratio of 0.6:1 was used as an example.

[0079] The vaccine composition of the present invention may additionally include an immunoadjuvant, but this is not essential, and sufficient vaccine effect is exhibited even without the immunoadjuvant. The immunoadjuvant that can be used in the present invention is characterized by being an immunoadjuvant selected from the group consisting of a group of substances that correspond to a pathogen-associated molecular pattern (PAMP) and react with a pattern recognition receptor (PRR), CpG DNA, lipoprotein, flagella, poly I:C, saponin, squalene, tricaprin, 3D-MPL, and detoxified lipooligosaccharide (dLOS), but is not limited thereto.

[0080] In the above-mentioned immune enhancer, the non-toxic lipooligosaccharide (detoxied lipooligosaccharide, dLOS) may be a substance disclosed in Korean Patent No. 1509456 or Korean Patent No. 2042993, but is not limited thereto.

[0081] As used herein, the term "lipid nanoparticle," also referred to as LNP, refers to a particle having at least one dimension of about a nanometer (e.g., 1 to 1,000 nm) comprising one or more lipids. In some embodiments, such lipid nanoparticles comprise a cationic lipid and one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. In some embodiments, mRNA or a portion thereof is encapsulated in the lipid portion of the lipid nanoparticle, or in an aqueous space surrounded by some or all of the lipid portion of the lipid nanoparticle, to protect the mRNA or portion thereof from enzymatic degradation or other undesirable effects induced by mechanisms of the host organism or cell, such as an adverse immune response. In some embodiments, the mRNA or portion thereof is associated with the lipid nanoparticle.

[0082] In the context of the present invention, lipid nanoparticles are not limited to any particular form, and should be construed to include any form formed when a cationic lipid or an ionic lipid, and optionally one or more additional lipids, are combined in an aqueous environment and / or in the presence of a nucleic acid compound. For example, liposomes, lipid complexes, lipoplexes, etc. are included within the scope of lipid nanoparticles.

[0083] In various embodiments, the lipid nanoparticles have a size of from about 30 nm to about 400 nm, from about 50 nm to about 400 nm, from about 70 nm to about 400 nm, from about 90 nm to about 400 nm, from about 110 nm to about 400 nm, from about 130 nm to about 400 nm, from about 150 nm to about 400 nm, from about 200 nm to about 400 nm, from about 250 nm to about 400 nm, from about 300 nm to about 400 nm, from about 350 nm to about 400 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or from about 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, The lipid nanoparticles have an average diameter of 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370 nm, 380 nm, 390 nm, 400 nm and are substantially non-toxic. In some embodiments, the mRNA, when present in the lipid nanoparticles, resists degradation by nucleases in aqueous solution. As used herein, the average diameter may be expressed as the z-average value determined by dynamic light scattering.

[0084] LNPs can comprise any lipid capable of forming particles to which one or more nucleic acid molecules are attached or in which one or more nucleic acid molecules are encapsulated. The term "lipid" refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters) and are generally characterized as being insoluble in water but soluble in various organic solvents. Lipids are usually divided into at least three classes: (1) "simple lipids," which include fats and oils as well as waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0085] LNPs comprising mRNA comprise one or more cationic lipids and one or more stabilizing lipids as defined herein. The stabilizing lipids include neutral lipids and PEGylated lipids.

[0086] LNPs comprise a cationic lipid. The cationic lipid preferably has cationizable properties. That is, the cationic lipid becomes protonated as the pH decreases below the pKa of the ionizable group of the lipid, but becomes progressively more neutral at higher pH values. When positively charged, the lipid can associate with negatively charged nucleic acids. In some embodiments, the cationic lipid comprises a zwitterionic lipid, which acquires a positive charge upon decreasing pH. The LNP can comprise any lipid capable of forming a particle to which one or more nucleic acid molecules are attached or encapsulated.

[0087] In some embodiments, the LNPs may comprise any additional cationic or cationizable lipid, i.e., any of a number of lipid species that possess a net positive charge at a selective pH, such as physiological pH.

[0088] The present invention also provides a method for producing a vaccine composition for preventing SARS-CoV-2 according to the present invention.

[0089] The method for producing a SARS-CoV-2 preventive vaccine composition according to the present invention is characterized by adding a solution or buffer containing liposomes or lipid nanoparticles to mRNA encoding the S antigen of the SARS-CoV-2 virus or a solution or buffer containing the same.

[0090] The mRNA, liposome, or lipid nanoparticle encoding the S antigen of the SARS-CoV-2 virus may be provided in the form of a lyophilized powder, or may be provided dissolved in a suitable solution or buffer. When the mRNA, liposome, or lipid nanoparticle encoding the S antigen of the SARS-CoV-2 virus is provided in a lyophilized state, the mRNA, liposome, or lipid nanoparticle may be dissolved in a suitable solution or buffer, and then a solution or buffer containing the liposome or lipid nanoparticle may be added to the mRNA encoding the S antigen of the SARS-CoV-2 virus or the solution or buffer containing the same, thereby producing a vaccine composition for preventing SARS-CoV-2 according to the present invention.

[0091] In addition, when dLOS is included in the vaccine composition encoding the S antigen of the SARS-CoV-2 virus according to the present invention, the SARS-CoV-2 preventive vaccine composition according to the present invention can be manufactured by adding mRNA encoding the S antigen of the ARS-CoV-2 virus or a solution or buffer containing the same to dLOS or a solution or buffer containing the same, and adding a solution or buffer containing liposomes or lipid nanoparticles.

[0092] dLOS may be provided in lyophilized powder form or dissolved in a suitable solution or buffer. If dLOS is provided in a lyophilized form, it may be used by dissolving it in a suitable solution or buffer.

[0093] In one embodiment of the present invention, it was confirmed that F-EG-COVID, a freeze-dried formulation of EG-COVID, a SARS-CoV-2 preventive vaccine according to the present invention, exhibited excellent immunogenicity even after a storage period of 8 weeks at -2 to 8 degrees Celsius (Fig. 7).

[0094] The cationic liposome used in the present invention is known to have a depot effect (Therapeutic Advances in Vaccines 2(6):159-82), and in another embodiment of the present invention, EG-COVID, a SARS-CoV-2 preventive vaccine using cationic liposomes as a carrier, was administered to the left thigh muscle of a rat, and CV-SF-614G mRNA expression in the serum and each tissue of the rat was confirmed over time, and it was confirmed that CV-SF-614G mRNA was expressed only in the administered area even over time (Fig. 8).

[0095] In another aspect of the present invention, the effect of inhibiting SARS-CoV-2 infection in Vero 76 cells was confirmed using the serum of mice administered EG-COVID, a vaccine for preventing SARS-CoV-2, and it was confirmed that the effect of inhibiting infection increased as the mRNA content of EG-COVID increased (Fig. 9A).

[0096] In another aspect of the present invention, it was confirmed that the EG-COVID of the present invention also exhibits excellent cross-immunity effects against SARS-CoV-2 mutants, namely alpha mutants and beta mutants (Fig. 9B).

[0097] In another aspect, the present invention relates to a method for preventing SARS-CoV-2 infection, comprising administering a composition for preventing SARS-CoV-2 comprising mRNA encoding the S antigen of the SARS-CoV-2 virus.

[0098] In another aspect, the present invention relates to the use of a composition comprising mRNA encoding the S antigen of the SARS-CoV-2 virus for the prevention of SARS-CoV-2 infection.

[0099] In another aspect, the present invention relates to the use of a composition for preventing SARS-CoV-2 infection comprising mRNA encoding the S antigen of the SARS-CoV-2 virus for the manufacture of a medicament for preventing SARS-CoV-2 infection.

[0100]

[0101] Example

[0102] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0103]

[0104] Example 1: Selection of mRNA sequences

[0105] The mRNA sequence was used to encode a sequence (CV-SF-614Gm) in which 986 (K) and 987 (V) were substituted with proline to stabilize the spike protein structure, and RRAR, an amino acid sequence from 682 to 685, was mutated to QQAQ.

[0106] Sequence optimization was performed using the following three programs to increase the content of guanine and cytosine in mRNA for the SARS-CoV-2 spike protein and the spike protein sequence of the SARS-CoV-2 614G variant, thereby stabilizing mRNA and increasing translation efficiency in humans.

[0107] Program1: GenSmart codon optimization program (Genscript)

[0108] Program2: Integrated DNA technologies codon optimization program (IDT, Integrated DNA Technologies)

[0109] Program3: GenArt codon optimization program (Thermo Fisher)

[0110]

[0111] Among the indicators that can predict RNA stability in detail, RNA fold, RNA fold thermodynamic ensemble, RNA structure, and thermodynamic energy of cofold were confirmed. In general, it is known that the lower the △G, the more thermodynamically stable it is. Therefore, CV-SF-WT-614D (mRNA encoding SARS-CoV-2 spike protein, SEQ ID NO: 1) and CV-SF-614Gm (mRNA encoding the spike protein of SARS-CoV-2 614G variant, SEQ ID NO: 2) were selected as the sequences derived from Program 3 with the lowest △G value.

[0112] The selected mRNA sequences were synthesized through in vitro transcription by TriLink BioTechnologies.

[0113] The DNA sequences of CV-SF-WT-614D and CV-SF-614Gm are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and the amino acid sequences of CV-SF-WT-614D and CV-SF-614Gm are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0114]

[0115]

[0116]

[0117] Example 2: Preparation of liposome delivery vehicle using film method

[0118] DOTAP (Merck & Cie / CH2900014), DOPE (Avanti Polar Lipid), and cholesterol (Avanti Polar Lipid) were mixed with chloroform in a ratio of 40:40:20, and completely dissolved at 37°C for 10 minutes to prepare a liquid solution.

[0119] The above liquid solutions were mixed in a round bottom flask at a certain weight ratio to create a lipid mixture, and the lipid mixture containing DOTAP was volatilized at 60°C for 30 minutes in a rotary evaporator (Buchi / B491_R200) ​​to evaporate chloroform and create a lipid membrane film on the flask wall.

[0120] Liposomes were formed by dissolving the lipid membrane in a flask containing 20 mM HEPES buffer (pH 7.4) containing 4% (w / v) sucrose at 60°C. The formed liposomes were measured for particle size, zeta potential, and dispersity using a dynamic light scattering analyzer. The remaining prepared liposomes were stored in a refrigerator at 4°C until testing.

[0121]

[0122] Example 3: In vitro expression confirmation according to the lipid composition of liposomes

[0123] A study has reported that when the Phosphatidylcholine (hereinafter □PC□) series lipid is changed to the Phosphoethanolamine (hereinafter □PE□) series lipid in liposomes, the amount of Erythropoietin (hereinafter □EPO□) protein expression increases by approximately 7 times (Dowhan Wet al., New Comprehensive Biochemistry, 36(4): 1-35, 2002; Leung A-KK et al., The journal of physical chemistry B, 119(28): 8698-8706, 2015; Kauffman KJ et al., Nano letters, 15(11):7300-7306, 2015).

[0124] In order to explore the optimal liposome composition to increase the delivery efficiency of mRNA, the optimal conditions for increasing the delivery efficiency of mRNA in vivo were explored using liposomes of the following four compositions, such as adding cholesterol (Chol) to DOTAP / DMPC, a constituent lipid of cationic liposomes used in the conventional shingles vaccine (EG-HZ), or replacing DMPC with 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (hereinafter □DOPE□).

[0125]

[0126] Group 1: DOTAP / DMPC (50:50, w / w)

[0127] Group 2: DOTAP / DMPC / Chol (40:40:20, w / w / w)

[0128] Group 3: DOTAP / DOPE (50:50, w / w)

[0129] Group 4: DOTAP / DOPE / Chol (40:40:20, w / w / w)

[0130]

[0131] Liposomes composed of the above different lipids were prepared, mixed with 20 μg of Renilla luciferase (TriLink BioTechnologies) and 80 μg of liposomes, and then a total of 100 μL was intramuscularly administered to 6-week-old female C57BL / 6N mice [Japan SLC] (Central Laboratory Animals) (n=4~5 / group). 6 hours after administration, the mice were anesthetized by intraperitoneal administration of 250 mg / kg of Avertin working solution, and Renilla luciferase substrate stock solution (0.37 mg / vial) was adjusted to 0.15 mg / mL by adding 2.4 mL of 1X PBS, and then intravenously administered at 1 mg / kg. Immediately after substrate administration, the mice were placed in an In vivo imaging system (IVIS) (Ami HTX) and images were taken with the exposure time set to 60 seconds, and the Region of interest (hereinafter referred to as □ROI□) values ​​of the injection site were compared.

[0132] As a result, as shown in Fig. 2, it was confirmed that cationic liposomes mixed with DOTAP / DOPE / Chol in a ratio of 40:40:20 (w / w / w) had the highest delivery efficiency in the body, and this was named CV-LP-b1 and used as an mRNA delivery system for EG-COVID.

[0133]

[0134] Example 4: Liposome Characterization

[0135] To develop EG-COVID, a vaccine to prevent SARS-CoV-2 virus infection, the physical properties of each of the three batches of CV-LP-b1, the mRNA vector identified in Example 3, were analyzed.

[0136] Dynamic light scattering (DLS) analysis was performed on liposomes CV-LP-b1 mixed in 20 mM HEPES buffer (pH 7.4) containing 4% sucrose using Malvern / ZSP to derive the average and standard deviation of particle size, dispersity, and zeta potential.

[0137] As a result, the average particle size of CV-LP-b1 was measured to be 80.3 ± 3.0 d. nm, the dispersity was 0.194 ± 0.010, and the zeta potential was 50.7 ± 3.0 mV.

[0138] Considering that the size of a typical liposome is 50 - 250 d. nm (Korean Patent Publication No. 2014-0097215), the size of the liposome was at an appropriate level, and it is known that when the zeta potential of the liposome is positively charged and is higher than 30 mV, aggregation does not occur and the structure is stably maintained (Antisense drug technology; Principles, Strategy, and Application, CRC press, second edition, 253, 2007), and since the zeta potential of the liposome was analyzed to be higher than that, it can be inferred that it is electrostatically stable. In addition, since the PDI of the liposome was all shown to be 0.25 or less, it was confirmed that the liposome had a particle distribution close to monodispersion in a stable state (Appl. Chem. Eng., 28 (2): 177-185, 2017).

[0139]

[0140] Example 5. Confirmation of NP ratio

[0141] The liposomes (hereinafter referred to as LP) and mRNA prepared in Examples 1 and 2 were mixed and adsorbed as main components in 20 mM HEPES (pH 7.4) containing 4% sucrose to prepare an mRNA liposome complex.

[0142] The N / P ratio, which is the mixing ratio of liposomes and mRNA, was calculated using the following formula, and the manufactured complexes were the EGFP mRNA-liposome complex of sequence number 8, the RLuc mRNA-liposome complex of sequence number 7, and the mRNA-liposome complex of CV-SF-614Gm of sequence number 2, which were used in DLS, in vivo expression, and immunogenicity experiments, respectively.

[0143]

[0144] mRNA-liposome complexes were prepared by varying the ratio of NP to mRNA forming a complex with liposomes, and in vivo expression of the mRNA-liposome complexes was confirmed using mice.

[0145] An mRNA complex expressing Rluc was intramuscularly injected into the deltoid muscle of 6-week-old female mice (C57BL / 6N, Orient Bio, Central Laboratory Animal) at a dose of 100 μl.

[0146] Six hours after administration of the test substance, the mice were anesthetized by intraperitoneal administration of Avertin working solution at 250 mg / kg, and Renilla luciferase substrate stock solution (0.37 mg / vial, Promega) was mixed with 2.4 mL of 1X PBS to make 0.15 mg / mL, and then intravenously injected at 1 mg / kg. Immediately after administration, the mice were photographed (xenogen IVIS-200) using Ami-HTX (Spectral Instruments Imaging, USA) with an exposure time set to 60 seconds, and the degree of expression at the injection site was quantified using Aura Imaging Software (Spectral Instruments Imaging, USA).

[0147] As a result, as shown in Fig. 3, mRNA expression efficiency in mice increased when the NP ratio was 0.23 or higher, and the highest expression was found at 0.46:1 to 1.0:1. In the following experiment, the mixing ratio of mRNA and liposomes was set to NP ratio = 0.6 and the experiment was conducted.

[0148]

[0149] Example 6. In vitro Expression by Selected Liposomes

[0150] In vitro expression was confirmed for a formulation (hereinafter, EG-COVID) in which CV-SF-614Gm was mixed with CV-LP-b1 to confirm whether normal expression occurred.

[0151] HEK293T cells (Homo sapiens embryonic kidney 293T cells, CRL-3216 / ATCC) were seeded in 100 mm dishes at 2 X 106 cells / dish, cultured overnight in a CO2 incubator at 37°C, and transfection was performed when the cells reached 60-70% confluency.

[0152] For transfection of CV-SF-614Gm, lipofectamine 3000 (Thermo Fisher) and CV-LP-b1 were mixed and treated in cells at 37°C in a CO2 incubator for 24 hours.

[0153] After 24 hours, all cell culture medium was removed, washed with D-PBS, and 400 μL of cell lysis solution 1X was dispensed into a 100 mm culture dish on ice. The cells were collected in a 1.7 mL tube using a scraper, vortexed, and centrifuged at 15,000 rpm, 4°C, for 15 minutes to collect the proteins in the supernatant. The collected proteins were quantified, and SDS-PAGE was performed on 20 μg / well of protein, and Western blotting was performed.

[0154] The antibodies used for Western blot are as follows.

[0155] SARS-CoV-2 antibody [NB100-56578 / Novus biologicals / ab092903c-15, (immunogen; SARS-CoV-2, amino acid 1124-1140 from S2 protein)]

[0156] SARS-CoV-2 antibody [MBS434243 / Mybiosource / T1218EL, (immunogen; SARS-CoV-2 S-full protein)]

[0157] SARS-CoV-2 antibody [40591-MM42 / Sino biological / HA14AP3001, (immunogen; SARS-CoV-2 S1-mFC protein)]

[0158] β-actin rabbit mAb HRP conjugate [5125 / Cell signaling / 6]

[0159] Goat anti-rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, HRP [G21234 / Thermo / 2087715]

[0160] Goat anti-mouse IgG secondary antibody, HRP [SSA007 / Sino biological / HO21OC1101]

[0161] As a result, as shown in Fig. 4A, it was confirmed that expression was concentration-dependent according to the CV-SF-614Gm concentration using lipofectamine 3000 (Thermo Fisher). In addition, as shown in Fig. 4B, when expression was observed by EG-COVID composed of different amounts of CV-SF-614Gm, it was confirmed that the S protein was expressed normally.

[0162]

[0163] Example 8. Confirmation of immunogenicity according to mRNA (CV-SF-614Gm) dose

[0164] Six-week-old, female, B6C3F1 / slc mice (central experimental animals) were administered two doses of 0.1 HD (human dose) mixed with different mRNA doses in CV-LP-b1 at three-week intervals. Two weeks after the final immunization, the mice were sacrificed, serum was isolated, and the SARS-CoV-2 receptor binding domain (RBD) protein-specific total IgG antibody titer (log10) was analyzed using an indirect ELISA method to derive the endpoint titer.

[0165]

[0166] To confirm the immunogenicity of the mRNA-liposome complex, 6-week-old female B6C3F1 / slc mice (Japan SLC) were selected as the target animals, and different mRNA doses were mixed with CV-LP-b1 and administered to the mice twice at 0.1 HD (human dose) at 3-week intervals. Then, 2 weeks after the final immunization, the mice were sacrificed, serum was separated, and the SARS-CoV-2 receptor binding domain (RBD) protein-specific total IgG antibody titer (log10) was analyzed using an indirect ELISA method to derive the endpoint titer.

[0167]

[0168] 8-1: Blood collection

[0169] Two weeks after the last administration, mice were anesthetized by intraperitoneal administration of 250 mg / kg of avertin working solution, and whole blood was collected by cardiac puncture. The collected whole blood was transferred to a microtube, allowed to stand at room temperature for 3 hours, and then centrifuged at 4°C and 15,000 rpm for 10 minutes. The supernatant was transferred to a new microtube to secure serum, which was then stored at -20°C until analysis.

[0170]

[0171] 8-2: Splenocyte restimulation

[0172] Mice were sacrificed by cervical dislocation, and the spleens were removed. The spleens from each group were pooled and transferred to a 24-well plate containing 1% penicillin-streptomycin solution (PBS w / antibiotics) (hereinafter, PBS w / antibiotics). The media used are shown in Tables 3 and 4.

[0173]

[0174]

[0175]

[0176]

[0177] Spleen tissue was washed with PBS containing antibiotics in a clean bench, transferred to a 60 mm dish containing 3 mL of basal media, and mashed with a 40 μm cell strainer to isolate splenocytes. The isolated splenocytes were transferred to a 15 mL tube and centrifuged at 4 °C and 3,000 rpm for 5 minutes. The supernatant was removed, and the splenocytes were treated with 3 mL of RBC lysis buffer (Thermo Fisher), left at room temperature for 3 minutes, and then centrifuged at 4 °C and 3,000 rpm for 5 minutes.

[0178] The supernatant was removed, the cells were suspended in 3 mL of PBS containing antibiotics, centrifuged at 4°C and 3,000 rpm for 5 minutes, the supernatant was removed, and the cells were suspended in 10 mL of complete media (Gibco).

[0179] The above cell suspension was cultured in complete medium at 2 x 10 7 After diluting to cells / mL, it was dispensed into a 96-well cell culture plate at 100 μl / well.

[0180] PepMix SARS-CoV-2-S1 peptide pool (JPT Peptide Technologies) and S2 peptide pool (JPT Peptide Technologies) were dissolved in 50 μL of DMSO per vial, and then mixed with complete medium to a final concentration of 2.5 μg / mL to prepare SARS CoV-2 spike peptide stimulant.

[0181] The stimulant (40 μg / well) and complete medium (60 μl / well) were added to 96 wells containing the cell suspension, and the reaction was performed for 72 hours under conditions of 37°C and 5% CO2.

[0182]

[0183] 8-3: Antibody analysis

[0184] RBD antigen (Mybiosource, USA) was diluted to 1 μg / mL using 1X PBS, dispensed 100 μl onto each immunoplate, covered with sealing film, and left in a refrigerator at 4°C overnight. 1 L of 1X PBS was prepared by diluting 20X PBS with purified water, and 500 μl of tween20 was added to prepare a washing buffer. The solution in each well was removed using an ELISA washer (Tecan / Hydroflexelisa) and washed 5 times using the washing buffer.

[0185] 1 g of BSA was dissolved in 100 mL of PBS to prepare a reagent diluent (1% BSA), and 200 μl / well was dispensed onto the immunoplate, covered with a sealing film, and left to stand in a 37 °C reactor for 1 hour. The solution in each well was removed with an ELISA washer, and washed five times with a washing buffer. 100 μl / well of the reagent diluent was dispensed onto the immunoplate. The serum sample obtained by method 6-1 was diluted 1:50 using the reagent diluent, and 100 μl was dispensed into row 1 of B to G of the immunoplate, and the sample was mixed by pipetting several times within the well, and then 100 μl was taken from row 1 and placed into row 2, thereby performing a 1 / 2 serial dilution of the sample up to row 12 on the ELISA plate.

[0186] To assess the suitability of the test, hyperserum was diluted 1:200 using a reagent diluent, dispensed as 100 μl into row 1 of each immunoplate H, and serially diluted 1 / 2 in the same manner as above.

[0187] The immunoplate was covered with sealing film and incubated in a 37°C reactor for 2 hours. The solution in each well was removed using an ELISA washer and washed five times with washing buffer.

[0188] Goat anti-mouse IgG antibody (Jackson Laboratory) was diluted 1:5,000 using a reagent diluent, dispensed 100 μl per immunoplate, covered with a sealing film, and reacted in a 37°C reactor for 1 hour.

[0189] The solution in each well was removed with an ELISA washer and washed five times with washing buffer. 100 μl of TMB substrate solution equilibrated to room temperature was dispensed onto each immunoplate and incubated in the dark at room temperature for 5 minutes. 100 μl of 1 N H2SO4 solution was dispensed onto each immunoplate to stop the reaction, and the absorbance was measured at 450 nm using an ELISA reader (Biotek / Epoch).

[0190]

[0191] 8-4: Cytokine analysis (ELISA)

[0192] The following experiment was conducted using an IFN-γ ELISA kit (Mouse IFN-γDuoset ELISA, R&D systems).

[0193] The culture medium of the splenocytes stimulated by the splenocyte re-stimulation method of 6-2 above was diluted 1 / 5 with a reagent diluent, dispensed at 100 μl / well onto a microplate coated with anti-mouse IFN-γ capture antibody (Jackson), covered with a sealing film, and left to stand at room temperature for 2 hours. Then, the solution in each well was removed with an ELISA washer (Tecan / Hydroflexelisa) and washed three times using a washing buffer.

[0194] Streptavidin-HRP in the kit was diluted 1 / 40 using a reagent diluent, dispensed 100 μl / well onto an immunoplate, covered with a sealing film, and left to stand at room temperature for 20 minutes. The solution in each well was removed using an ELISA washer and washed three times using a washing buffer.

[0195] The anti-mouse IFN-γ detection antibody in the kit was diluted to 200 ng / mL using Reagent Diluent, dispensed 100 μL / well into the immunoplate, covered with sealing film, and left to stand at room temperature for 1 hour. The solution in each well was removed using an ELISA washer (Tecan / Hydroflexelisa), and washed three times using wash buffer.

[0196] 100 μl of TMB substrate (KPL sureblue TMB microwell peroxidase substrate, Seracare) solution was dispensed onto each immunoplate and reacted in a dark place at room temperature for 15 minutes. Then, 100 μl of 1N H2SO4 solution was dispensed onto each immunoplate to stop the reaction and the absorbance was measured at 450 nm using an ELISA reader.

[0197]

[0198] 8-5 Neutralization

[0199] The SARS-CoV-2 surrogate virus neutralization test (sVNT) Kit (Genscript) was used to test serum samples obtained by administering the EG-COVID vaccine of the present invention to determine whether the vaccine effectively inhibits viral infection.

[0200] The negative control, positive control, 60 μL of serum sample, and 60 μL of 1:1000 diluted-HRP conjugated RBD were mixed in a 1.5 mL microtube, reacted in a 37 °C incubator for 30 minutes, and 100 μL was dispensed into a microtiter test strip plate, covered with a sealing film, and reacted in a 37 °C incubator for 15 minutes.

[0201] The solution in each well was removed using an ELISA washer (Tecan / Hydroflexelisa) and washed 4 times using 1X wash solution.

[0202] TMB solution (Thermo Fisher) was dispensed at 100 μL / well, covered with sealing film, and reacted for 15 minutes in a dark place at room temperature. Then, stop solution was dispensed at 50 μL / well to stop the reaction, and the optical density was measured at 405 nm using an ELISA reader (Thermo Scientific). The neutralizing antibody formation ability (Neutralization%) was quantified as follows.

[0203] Figure 5A shows the antibody titer of RBD-specific IgG analyzed by ELISA in the serum of mice administered the mRNA complex of CV-SF-614Gm mixed with CV-LP-b1, and it was confirmed that the transfer ability by adsorption was excellent up to 30 μg mRNA, Figure 5B shows the IFN-γ concentration analyzed by ELISA in the serum of mice administered the mRNA complex of CV-SF-614Gm mixed with CV-LP-b1, and high IFN-γ concentrations were observed at 5 μg and 10 μg mRNA, and Figure 5C shows the neutralizing antibody formation ability analyzed using the SARS-CoV-2 surrogate virus neutralization test (sVNT) Kit in the obtained serum, and high immune-inducing ability was observed at 5 μg or more of mRNA.

[0204] When the above results were converted to HD (human dose), CV-LP-b1 was confirmed to have excellent delivery ability through adsorption up to 300 μg mRNA, and it was determined that mRNA of 50 μg or more was appropriate for immune induction ability. However, in terms of cellular immunity as viewed by IFN-gamma concentration, it was found to be considerably excellent at 5 to 10 μg.

[0205]

[0206] Example 9: Differences in immunogenicity according to the number of administrations

[0207] 200 μg of CV-SF-614Gm was mixed with CV-LP-b1 at an NP ratio of 0.6:1, lyophilized, and the difference in immunogenicity between single and double administration using 0.1 HD was confirmed.

[0208] B6C3F1 / slc, female (central experimental animal), was immunized twice at 3-week intervals for 6 weeks, sacrificed 2 weeks later, or sacrificed 2 weeks after a single administration, and immunogenicity was confirmed using the obtained serum and splenocytes (n=6 / group).

[0209] The experimental method was performed in the same manner as in Example 8, and the results are shown in Figure 6. It was confirmed that immunogenicity, including humoral and cellular properties, was superior when administered twice than when administered once.

[0210]

[0211] Example 10. Confirmation of stability of freeze-dried formulation

[0212] The COVID-19 vaccines currently under development, mRNA-based vaccines from Pfizer and Moderna, use lipid nanoparticles (hereinafter referred to as □LNP□) as mRNA carriers, which means they have poor refrigerated storage stability and require distribution under ultra-low temperature storage conditions (-20℃ to -80℃).

[0213] On the other hand, EG-COVID uses cationic liposomes, and in this example, the in vivo efficacy of L-EG-COVID, a liquid formulation of the cationic liposome CV-LP-b1 and CV-SF-614Gm complex (EG-COVID) stored in a refrigerator for 8 weeks, and F-EG-COVID, a lyophilized formulation, was confirmed, and the immunogenicity of the finished product stored in a refrigerator for 0 and 4 weeks was compared and analyzed, thereby comparing the in vivo efficacy stability according to the refrigerator storage period for each formulation in the same manner as in Example 8.

[0214] In this example, EG-COVID was manufactured to contain 100 μg of CV-SF-614Gm, and after a storage period at -2 to 8°C for a certain period of time for the liquid formulation or lyophilized formulation, the lyophilized formulation was rehydrated and an immunogenicity test was conducted to confirm that the efficacy was maintained.

[0215]

[0216] A freeze-dried formulation was prepared using the liquid formulation prepared in Example 2 using the following method.

[0217] The liquid EG-COVID formulation was dispensed in 0.65 ml portions into sterilized 2 ml glass vials, half-closed with a rubber stopper, transferred to a freeze dryer (Ilshin Biobase), and freeze-dried in the following order: -40°C (50 mTorr) for 10 hours, -20°C (50 mTorr) for 10 hours, and 20°C (50 mTorr) for 20 hours.

[0218] The vials after lyophilization were completely sealed with rubber stoppers and stored at 2-8°C.

[0219] As a result, as shown in Fig. 7, the liquid formulation was found to have severely reduced stability after 4 weeks of storage and was unable to induce immunogenicity, but the lyophilized formulation was found to stably maintain immunogenicity for up to 8 weeks, confirming that the lyophilized formulation of EG-COVID has excellent storage stability.

[0220]

[0221] Example 11. Biodistribution

[0222] The COVID-19 vaccine EG-COVID according to the present invention utilizes cationic liposomes as mRNA delivery vehicles to efficiently deliver CV-SF-614Gm into the body. In a previous study by the inventors, a mixture of mRNA encoding Renilla luciferase and CV-LP-b1 was administered intramuscularly to mice. The results of this study showed that protein expression occurred only at the injection site from 6 to 24 hours after administration, with no expression in other organs.

[0223] In this example, we administered EG-COVID intramuscularly to rats and observed the distribution of CV-SF-614Gm at the injection site and in major organs outside the injection site over time. To this end, we measured the relative value of CV-SF-614Gm compared to GAPDH, a housekeeping gene in rats, using RT-qPCR to determine the body distribution of CV-SF-615Gm after EG-COVID administration.

[0224] In this example, to confirm the body distribution pattern of CV-SF-614Gm over time, EG-COVID was administered to the left thigh muscle of rats, and then the presence of CV-SF-614Gm in the body was confirmed through RT-qPCR after 0, 2, 6, 24, 48, 72, and 120 hours, respectively.

[0225] 105 6-week-old male SD rats (Orient Bio) were used as experimental animals, and the test groups are shown in Table 5.

[0226]

[0227]

[0228]

[0229]

[0230] Serum and each tissue sample were obtained from the test group rats, and total RNA was extracted from the serum and tissue using the RNeasy Micro kit (QIAGEN / 74004) according to the test method recommended by the manufacturer. The concentration and yield of total RNA were confirmed using Nanodrop (Thermo Fisher).

[0231]

[0232]

[0233] A GAPDH primer and probe set (Thermo Fisher) was used with rat GAPDH as a reference gene, and the 5' end of the primer was tagged with VIC dye.

[0234]

[0235]

[0236] As shown in Tables 9 and 10 below, sample 1 (tissue-derived RNA), TaqPath 1-step multiplex master mix (hereinafter referred to as □master mix□, (Thermo Fisher / A28523)), primer & probe, GAPDH assay mix, and nuclease-free water were dispensed into a 96-well (Invitrogen / A28138) or 384-well plate (Invitrogen / A28140) using a multi-channel pipette to make a final volume of 15 μL, and qRT-PCR reaction was performed.

[0237]

[0238]

[0239]

[0240]

[0241] The reaction results for each sample were calculated as follows to determine the amount of CV-SF-614Gm relative to GAPDH.

[0242] CV-SF-614Gm Ct(△R) vs. GAPDH = Average Ct of CV-SF-614Gm - Average Ct of GAPDH

[0243] And, for accurate comparison, △R was converted to an exponent.

[0244]

[0245] Afterwards, the distribution pattern of CV-SF-614Gm in the body was analyzed by drawing a graph by group, time, and tissue.

[0246] As a result, the amount of CV-SF-614Gm compared to GAPDH was confirmed in 12 major organs and serum including the administration site, and CV-SF-614Gm was detected only in the left thigh muscle (ISL), which was the administration site.

[0247] The relative index of CV-SF-614Gm relative to GAPDH in ISL was observed to be up to 57% 2 hours after EG-COVID administration, and up to 70% 6 hours later. CV-SF-614Gm was detected at its highest level at 6 hours, gradually decreasing thereafter, and undetectable after 72 hours. During the observation period, CV-SF-614Gm was not detected in major organs other than the injection site (see Figures 8a and 8b).

[0248]

[0249] Example 12. Evaluation of neutralizing antibody titer (Plaque Reduction Neutralization test)

[0250] To evaluate the neutralizing antibody titer of EG-COVID and confirm its protective effect against SARS-CoV-2 (NCCP 43326, Wuhan-origin virus), the following experiment was conducted.

[0251] Six-week-old female B6C3F1 / slc mice (n=6 / group) (Orient Bio) were used as experimental animals. EG-COVID containing CV-SF-614Gm was administered twice at three-week intervals to the negative control and test groups, respectively, and blood samples were collected two weeks later.

[0252] The virus experiment was conducted at the National Masan Hospital, which has a BL3 facility.

[0253] 8 X 10 of Vero 76 cell line (ATCC CRL-1587™) were seeded in a 6-well plate. 5 After seeding cells / well, they were cultured for 24 hours to prepare for sub-confluence. The serum from the EG-COVID administration group and the serum from the negative control group were each diluted 1 / 10-fold in serum-free DMEM, and then serially diluted 2-fold up to 1 / 20480-fold, and 105 μL of the diluted serum was added to a concentration of 400 pfu / 10 5 After mixing 1:1 with μL of virus (SARS-CoV-2 (NCCP 43326)) solution, it was incubated at 37°C for 1 hour.

[0254] The culture medium of the Vero 76 cell line was removed and washed with PBS. 200 μL of the serum and virus mixture was added, shaken every 15 minutes, and cultured at 37°C for 90 minutes to allow infection. The culture medium was then removed and washed with PBS to remove uninfected virus. The cells were then covered with DMEM medium (Gibco) containing 2% FBS and 1% agarose and cultured in a 37°C incubator for 3 days. Upon completion of the incubation, a 4% formaldehyde solution was added to the agarose, fixed for 1 hour at room temperature, and the agarose was carefully removed. The fixed cell layer was stained with a 0.5% crystal violet (in 20% methanol) solution. After washing three times with PBS, the plates were completely dried. The crystal violet-stained infected cells (hereinafter referred to as "Plaque") were counted and compared with the unneutralized control group to derive the virus infection inhibition rate, as shown in the following formula.

[0255]

[0256] (The control group was the group that did not neutralize the virus)

[0257] Neutralizing antibody titers were derived as IC50 titers (log10) by calculating the dilution factor that reduces the virus infection inhibition rate by 50% using nonlinear regression in GraphPad Prism software.

[0258] As a result, as shown in Fig. 9A, for infection with SARS-CoV-2 wild type virus (NCCP43326, The National Culture Collection for Pathogens in Korea), the IC50 titer (log10) was 3.569 ± 0.418, 4.039 ± 0.357, and 4.375 ± 0.443 (mean ± standard deviation) in EG-COVID administration group 1 (2.5 μg / mouse), EG-COVID administration group 2 (5 μg / mouse), and EG-COVID administration group 3 (10 μg / mouse), respectively. It was confirmed that the neutralizing antibody titer tended to increase as the mRNA content increased.

[0259] Additionally, as shown in Figure 9B, it was confirmed that excellent cross-immunity was observed against SARS-CoV-2 variants, namely alpha variant and beta variant.

[0260]

[0261] The SARS-CoV-2 preventive vaccine according to the present invention not only exhibits excellent vaccine efficacy by exhibiting excellent stability and high immunogenicity in vivo without the need for additional immune boosters, but also maintains vaccine efficacy even when manufactured in a lyophilized form, making storage and use of the vaccine easy, and thus, excellent preventive efficacy against COVID-19 can be expected.

[0262]

[0263] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0264]

[0265] Electronic file attached.

Claims

1. A vaccine composition for preventing SARS-CoV-2 comprising mRNA encoding the S antigen of the SARS-CoV-2 virus.

2. A SARS-CoV-2 preventive vaccine composition according to claim 1, characterized in that the mRNA encoding the S antigen has a base sequence of sequence number 1 or sequence number 2.

3. A SARS-CoV-2 preventive vaccine composition according to claim 1 or 2, characterized in that it additionally contains liposomes or lipid nanoparticles.

4. A SARS-CoV-2 preventive vaccine composition according to claim 3, wherein the liposome or lipid nanoparticle comprises a cationic lipid.

5. A SARS-CoV-2 preventive vaccine composition according to claim 4, wherein the liposome or lipid nanoparticle further comprises a neutral lipid.

6. A SARS-CoV-2 preventive vaccine composition according to claim 4 or 5, characterized in that the liposome or lipid nanoparticle additionally contains cholesterol.

7. In any one of claims 4 to 6, the cationic lipid is dimethyldioctadecylammonium bromide (DDA), C12-200, 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 3β-[N-(N′,N′-dimethylaminoethane) carbamoyl cholesterol (DC-Chol), 1,2-dioleoyloxy-3-dimethylammoniumpropane (DODAP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1 Etyle PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:1 Ethyl PC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1 Ethyl PC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholin (18:0 Ethyl PC), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (16:0 Ethyl PC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0 Ethyl PC), 1,2-dilauroyl-sn-glycero-3-ethylphosphocholin (12:0 Ethyl PC),N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dimyristoyl-3-dimethylammonium-propane (14:0 DAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (16:0DAP), 1,2-distearoyl-3-dimethylammonium-propane (18:0 DAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 1,2-stearoyl-3-trimethylammonium-propane (18:0 TAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (16:0 TA), A vaccine composition for preventing SARS-CoV-2, characterized by at least one selected from the group consisting of 1,2-dimyristoyl-3-trimethylammonium-propane (14:0 TAP) and N4-cholesteryl-spermine (GL67).

8. In the fifth paragraph, the neutral lipid is 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), A SARS-CoV-2 preventive vaccine composition, characterized in that it comprises at least one selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), phosphatidylserine (PS), phosphoethanolamine (PE), phosphatidylglycerol (PG), phosphoric acid (PA), and phosphatidylcholine (PC), and 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol) (DOPI), 1,2-distearoyl-sn-glycero-3-phosphoinositol (DSPI).

9. A SARS-CoV-2 preventive vaccine composition according to any one of claims 5 to 8, wherein the weight ratio of the cationic lipid and the neutral lipid is 1:9 to 9.5:0.

5.

10. A SARS-CoV-2 preventive vaccine composition according to claim 6, characterized in that the weight ratio of the cationic lipid and cholesterol is 6:1 to 1:

3.

11. A SARS-CoV-2 preventive vaccine composition according to claim 10, characterized in that the weight ratio of the cationic lipid, neutral lipid, and cholesterol is 1 to 9.5: 0.5 to 9: 0.05 to 3.

12. A SARS-CoV-2 preventive vaccine composition according to claim 3, wherein the N:P ratio of the liposome or lipid nanoparticle and mRNA is 0.2:1 to 1.4:

1.

13. A SARS-CoV-2 preventive vaccine composition according to any one of claims 1 to 12, characterized in that it further comprises an immunostimulant.

14. A SARS-CoV-2 preventive vaccine composition according to claim 13, wherein the immune enhancer comprises at least one immune enhancer selected from the group consisting of PAMP, saponin, CpG DNA, lipoprotein, flagella, poly I:C, squalene, tricaprin, 3D-MPL, and detoxied lipooligosaccharide (dLOS).

15. A SARS-CoV-2 preventive vaccine composition according to any one of claims 1 to 14, characterized in that it is in the form of a lyophilized formulation.

16. A method for producing a vaccine composition for preventing SARS-CoV-2, characterized by adding a solution or buffer containing liposomes or lipid nanoparticles to mRNA encoding the S antigen of the SARS-CoV-2 virus or a solution or buffer containing the same.

Images