WO2023121264A1 - Vaccine composition for sars-cov-2 variant, and use thereof - Google Patents

Abstract

The present disclosure relates to a vaccine composition for preventing a SARS-CoV-2 variant, the composition comprising mRNA encoding an S antigen of a SARS-CoV-2 variant virus. A vaccine for preventing the SARS-CoV-2 variant according to the present disclosure exhibits excellent stability and high immunogenicity in vivo and is thus easy to store and use, and is expected to have an excellent preventive effect against COVID-19.

Description

Mutant SARS-COV-2 Vaccine Compositions and Uses Thereof

This disclosure was made by the project number HV20C0132 under the support of the Ministry of Health and Welfare of the Republic of Korea, and the specialized research management institution for the project is the Vaccine Commercialization Technology Development Center, the research project name is “infectious disease prevention and treatment technology development”, and the research project name is “quick response to pandemic” Development of a new mRNA-based vaccine platform that can effectively respond”, the leading institution is Igene Co., Ltd., and the research period is 2021.12.01-2023.06.30.

In addition, this disclosure was made by the project number HV22C0052 under the support of the Ministry of Health and Welfare of the Republic of Korea, and the research management specialized institution of the project is the Korea Health Industry Development Institute, the research project name is “Clinical Support for mRNA Vaccine for New Infectious Disease Response”, and the research project name is “Multivalent Development of mRNA vaccine to prevent COVID-19”, Hosted by I-Gene Co., Ltd., research period 2022.04.01-2023.03.31.

This patent application claims priority to Korean Patent Application No. 10-2021-0182914 filed with the Korean Intellectual Property Office on December 20, 2021, the disclosure of which is incorporated herein by reference.

The present disclosure relates to a mutant SARS-CoV-2 vaccine composition, and more particularly, to a mutant SARS-CoV-2 vaccine composition comprising mRNA encoding an S antigen of mutant SARS-CoV-2, and uses thereof.

Coronavirus is a type of RNA virus whose genetic information is composed of ribonucleic acid (RNA). It causes respiratory and digestive tract infections in humans and animals. Mainly, it is easily infected by mucosal transmission, droplet transmission, etc., and generally causes mild respiratory infections in humans, but rarely causes fatal infections.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), which is currently causing the pandemic, has been genetically sequenced (DNA sequencing). ) It is a positive sense single-stranded RNA coronavirus.

SARS-CoV-2 virus uses the spike protein on the surface to induce airway epithelial cells, alveolar epithelial cells, vascular endothelial cells, and macrophages in the lungs. (Macrophage) Invades into the host cell by binding to angiotensin-converting enzyme 2 (ACE2) present on the surface.

As a result of the genome sequence study of SARS-CoV-2, a receptor-binding domain (RBD) with a tertiary structure very similar to that of SARS-CoV was identified in the spike protein, and SARS-CoV It was speculated that the RBD of -2 has a higher affinity for ACE2 than the RBD of SARS-CoV, so that SARS-CoV-2 is more infectious than SARS-CoV.

The spike protein of SARS-CoV-2 is composed of two proteins, S1 and S2. Among them, the S1 protein is composed of an amino-terminal domain and an RBD. When RBD binds to ACE2, SARS-CoV-2 virion enters the endosome of the cell through endocytosis, and then the fusion peptide is exposed and breaks down the host cell membrane. inserted into The S2 protein consists of a fusion peptide region (FP region) and heptad repeat regions (HR1, HR2). When HR1 and HR2 touch each other, they fuse to the viral membrane, releasing SARS-CoV-2 virions out of the host cell. S1 and S2 have different cleavage sites and are cleaved by each protease, resulting in SARS-CoV-2 infection. SARS-CoV-2 treatments and vaccines are being developed using a strategy to disrupt the binding between protease 2 (TMPRSS2) and the virus.

People infected with SARS-CoV-2 may develop mild to severe symptoms, such as fever, cough, shortness of breath, and diarrhea. People with complications or illness, the elderly, are more likely to die. In particular, early detection and treatment are very important because those with underlying diseases such as heart disease and diabetes are more vulnerable to infections and suffer from complications or organ damage.

RNA viruses such as SARS-CoV-2 evolve with a high mutation frequency during genome replication, forming a 'lineage', a group of genetically closely related viral mutations derived from a common ancestor. SARS-CoV-2 has also formed a lineage through continuous mutations since the start of the pandemic, and among them, the regional range related to the mutation rate, the effect of the mutation group on medical measures, the severity of the disease, and the potential or Considering the known impact, it is managed under the SIG variant classification system.

The SIG taxonomy defines SARS-CoV-2 into four classes: variant under monitoring (VBM), variant of interest (VOI), variant of concern (VOC), and variant of high risk (VOHC).

Of these, the mutations of concern are delta (B.1.617.2 and AY lines) and omicron (B.1.1.529) discovered in November 2021. Other 10, including alpha (B.1.1.7 and Q strains), beta (B.1.351 and derivatives), gamma (P.1 and derivatives), and epsilon (B.1.427 and B1.429). Species Variation The virus is set to the variation being monitored.

The biggest problem with the mutant virus is that since a number of mutations occur in the spike protein, it is characterized by a decrease in neutralization efficacy by previously licensed vaccines, and in the case of delta, there is a case of becoming a dominant species due to improved infectivity ( Business Insider, May 2021).

The currently licensed vaccine is a vaccine manufactured based on the first virus-derived spike protein of SARS-CoV-2, and since the developer is also aware of the problem of reduced efficacy, we are responding with the development of a booster vaccine and a new vaccine to solve this problem. As such, there is still a need to develop a vaccine that exhibits high immunogenicity while maintaining its effect more stably.

A number of papers and patent documents are referenced throughout this specification and their citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the content of the present disclosure and the level of the technical field to which the present disclosure belongs.

The present inventors have made intensive research efforts to develop a preventive vaccine against mutant SARS-CoV-2, which has excellent storage stability and excellent immunogenicity in the body. As a result, the present invention was completed by developing an mRNA vaccine loaded with a nucleic acid encoding a mutant antigen for the spike protein of SARS-CoV-2, and confirming that the vaccine exhibits excellent stability and high immunogenicity.

Accordingly, an object of the present disclosure is to provide a variant SARS-CoV-2 vaccine composition that exhibits excellent stability and high immunogenicity.

Other objects and advantages of the present disclosure will become more apparent from the following detailed description of the invention, claims and drawings.

The present inventors have made intensive research efforts to develop a preventive vaccine against mutant SARS-CoV-2, which has excellent storage stability and excellent immunogenicity in the body. As a result, an mRNA vaccine loaded with a nucleic acid encoding a mutant antigen for the spike protein of SARS-CoV-2 was developed, and it was confirmed that the vaccine exhibits excellent stability and high immunogenicity.

The present disclosure relates to a variant SARS-CoV-2 vaccine composition.

Unless defined otherwise, 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 belongs. In general, the nomenclature used herein is one well known and commonly used in the art.

Hereinafter, the present disclosure will be described in more detail.

According to one aspect of the present disclosure, the present disclosure provides a variant SARS-CoV-2 vaccine composition comprising mRNA encoding the Spike antigen of severe acute respiratory syndrome coronavirus 2 variant (SARS-CoV-2).

In the present disclosure, 'mutated SARS-CoV-2' includes all SARS-CoV-2 mutations in the antigen of SARS-CoV-2, in particular, mutations such as the receptor binding domain (RBD) of the S antigen. used in the sense of

The shift is an Omicron shift.

According to one embodiment of the present disclosure, the mutant SARS-CoV-2 vaccine composition of the present disclosure is a multivalent vaccine that combines (mixes) a vaccine based on the mutation and/or a vaccine to prevent other infectious (infectious) diseases, if necessary. (multivalent) form can be prepared.

Accordingly, the mutant SARS-CoV-2 vaccine composition of the present disclosure can induce a more effective immune response by simultaneously protecting various types of infectious factors.

The Omicron mutation (lineage B.1.1.529) is A67V, Δ69-70, T95I, G142D/Δ143-145, Δ211/L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493K, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F mutations/ Mutations with deletions SARS-CoV- 2.

In the present disclosure, 'Spike antigen' or 'S antigen' refers to a spike protein that induces antibody production against SARS-CoV-2 and similar proteins (antigens) by modifications such as sequence alteration, fragmentation, or fusion thereof. used in the sense of inclusion.

The mRNA encoding the S antigen of the mutant SARS-CoV-2 may be the mRNA of SEQ ID NO: 1 encoding the Omicron-PQ SARS-CoV-2 S protein. The mRNA of SEQ ID NO: 1 may additionally encode an Omicron mutant S protein having a substituted sequence of K986P, V987P, or 682-QQAQ-685.

The S antigen of the mutant SARS-CoV-2 may include the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

The mRNA encoding the S antigen of the mutant SARS-CoV-2 may be an mRNA having at least one open reading frame that can be translated by a cell or organism, which is typically provided together with the mRNA. The product of this translation is an antigen, preferably a peptide or protein capable of serving as an immunogen. The product may also be a fusion protein consisting of two or more immunogens, for example a fusion protein consisting of two or more epitopes, peptides or proteins derived from the same or different viral proteins, wherein the epitopes, peptides or proteins comprise a linker sequence can be connected by

In addition, it can be understood that the mRNA encoding the S antigen of the mutant SARS-CoV-2 is an artificial mRNA, that is, a non-naturally occurring mRNA molecule. An artificial mRNA molecule can be understood as a non-natural mRNA molecule. Such mRNA molecules may be non-natural due to (non-naturally occurring) individual sequences and/or other non-naturally occurring alterations, such as structural alterations of nucleotides. Artificial mRNA molecules can be designed and/or produced by genetic engineering methods that correspond to a desired artificial sequence of nucleotides (heterologous sequence). That is, the mRNA encoding the mutant SARS-CoV-2 virus S antigen differs from the wild-type sequence in at least one nucleotide. A 'wild type' herein may be understood as a naturally occurring sequence.

In a specific embodiment of the present disclosure, uridine included in the mRNA of the present disclosure may be substituted with 5-methoxy-uridine.

Furthermore, the mRNA encoding the S antigen of the mutant SARS-CoV-2 is degraded in vivo (e.g., by exo- or endo-nuclease) and/or ex vivo (e.g., prepared prior to vaccine administration). process, for example in the preparation of vaccine solutions to be administered). Stabilization of the RNA can be achieved, for example, by providing a 5'-CAP structure, poly-A-tail, or any other UTR modification. Stabilization of RNA can also be achieved by chemical modification or modification of the G/C content of nucleic acids. A variety of other methods are known in the art and are applicable to the present disclosure.

In one embodiment of the present disclosure, the mRNA further includes a 5'UTR. The 5' UTR includes the sequence of SEQ ID NO: 3.

In one embodiment of the present disclosure, the mRNA further includes a 3' UTR. The 3' UTR includes the sequence of SEQ ID NO: 4.

In one embodiment of the present disclosure, the mRNA may include a 5'Cap structure. The 5' Cap is m7G(5')ppp(5')(2'OMeA)pG Cap, m7G(5')ppp(5')(2'OMeA)pU Cap, m7(3'OMeG)(5' )ppp(5')(2'OmeA)pG Cap, 3'-O-Me-m7G(5')ppp(5')G Cap, G(5')ppp(5')G Cap, or m7G( 5')ppp(5')G Cap may be, but is not limited thereto.

In one embodiment of the present disclosure, the mRNA further includes a poly A tail having a length of 50 to 150 nt. In specific embodiments, the poly A tail is 50 to 150 nt, 50 to 120 nt, 50 to 100 nt, 50 to 90 nt, 50 to 80 nt, 50 to 70 nt, 50 to 60 nt, 60 to 150 nt , 60 to 120 nt, 60 to 100 nt, 60 to 90 nt, 60 to 80 nt, 60 to 70 nt, 70 to 150 nt, 70 to 120 nt, 70 to 100 nt, 70 to 90 nt, 70 to 80 nt , 50 nt, 60 nt, 70 nt, 80 nt, 90 nt, or 100 nt, but is not limited thereto.

In one embodiment of the present disclosure, the mRNA may additionally include RNA having an ORF encoding a signal peptide fused to the S antigen of SARS-CoV-2.

A signal peptide comprising 10-60 amino acids at the N-terminus of a protein is typically required for transmembrane translocation on the secretory pathway and thus permits entry of most proteins into the secretory pathway in both eukaryotes and prokaryotes. universally controlled. In eukaryotes, the signal peptide of the nascent precursor protein (pre-protein) guides the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates transport of the growing peptide chain across the membrane for processing. ER processing results in mature proteins, and the signal peptides are typically cleaved from the precursor proteins by the host cell's ER-resident signal peptidases, or they remain uncleaved and function as membrane anchors. Signal peptides can also facilitate targeting of proteins to cell membranes.

Signal peptides can be 10-60 amino acids in length. For example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 signal peptides. 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 , 58, 59, or 60 amino acids in length.

In one embodiment of the present disclosure, the signal peptide includes the amino acid sequence of SEQ ID NO: 5.

In one embodiment of the present disclosure, the signal peptide comprising the amino acid sequence of SEQ ID NO: 5 may be encoded by mRNA represented by the nucleotide of SEQ ID NO: 6, but is not limited thereto.

In one embodiment of the present disclosure, cDNA encoding the polynucleotide can be transcribed using an in vitro transcription (IVT) system. In vitro transcription of RNA is known in the art and is described in International Publication No. WO 2014/152027, which is incorporated herein by reference in its entirety.

In some embodiments, RNA transcripts are generated using a non-amplified, linearized DNA template in an in vitro transcription reaction to generate RNA transcripts. In some embodiments, template DNA is isolated DNA. In some embodiments, template DNA is cDNA. In some embodiments, cDNA is formed by reverse transcription of RNA polynucleotides, such as but not limited to coronavirus mRNA. In some embodiments, a cell, eg, a bacterial cell, eg, E. coli, eg, a DH-1 cell, is transfected with a plasmid DNA template. In some embodiments, transfected cells are cultured to replicate plasmid DNA, which is then isolated and purified. In some embodiments, the DNA template is located 5' to the gene of interest and includes an operably linked RNA polymerase promoter, eg, the T7 promoter.

The variant SARS-CoV-2 vaccine composition of the present disclosure may further include liposomes or lipid nanoparticles (LNPs). Specifically, the mRNA encoding the S antigen of the mutant SARS-CoV-2 included in the vaccine composition of the present disclosure is adsorbed or associated with the outside of liposomes or lipid nanoparticles, or encapsulated or encapsulated inside. may be in a state

In the present disclosure, 'liposome' refers to a material used to stably deliver physiologically active components (eg, mRNA) and maximize the penetration effect.

In the present disclosure, a 'lipid nanoparticle' refers to a particle having at least one dimension on the order of nanometers (eg, 1 to 1,000 nm), comprising one or more lipids. Such lipid nanoparticles may contain one or more excipients selected from cationic lipids, neutral lipids, charged lipids, steroids and polymer conjugated lipids.

The lipid nanoparticle is not limited to any particular form, and is produced when, for example, 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. should be construed to include any form. For example, lipid complexes, lipoplexes, etc. are within the scope of lipid nanoparticles.

The lipid nanoparticles are about 30 nm to about 200 nm, 40 nm to about 200 nm, about 50 nm to about 200 nm, about 60 nm to about 200 nm, about 70 nm to about 200 nm, about 80 nm to about 200 nm. nm, about 90 nm to about 200 nm, about 100 nm to about 200 nm, about 110 nm to about 200 nm, about 120 nm to about 200 nm, about 130 nm to about 200 nm, about 140 nm to about 200 nm, About 150 nm to about 200 nm, about 30 nm to about 180 nm, about 40 nm to about 180 nm, about 50 nm to about 180 nm, about 60 nm to about 180 nm, about 70 nm to about 180 nm, about 80 nm to about 180 nm, about 90 nm to about 180 nm, about 100 nm to about 180 nm, about 110 nm to about 180 nm, about 120 nm to about 180 nm, about 130 nm to about 180 nm, about 140 nm to About 180 nm, about 30 nm to about 160 nm, about 40 nm to about 160 nm, about 50 nm to about 160 nm, about 60 nm to about 160 nm, about 70 nm to about 160 nm, about 80 nm to about 160 nm nm, about 90 nm to about 160 nm, about 100 nm to about 160 nm, about 110 nm to about 160 nm, about 120 nm to about 160 nm, about 130 nm to about 160 nm, about 140 nm to about 160 nm, About 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 150 nm, about 70 nm to about 150 nm, about 80 nm to about 150 nm, about 90 nm nm to about 150 nm, about 100 nm to about 150 nm, about 110 nm to about 150 nm, about 120 nm to about 150 nm, about 130 nm to about 150 nm, about 140 nm to about 150 nm, about 30 nm to About 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 150 nm, about 70 nm to about 150 nm, about 80 nm to about 150 nm, about 90 nm to about 150 nm nm, about 100 nm to about 150 nm, about 110 nm to about 150 nm, about 120 nm to about 150 nm, about 130 nm to about 150 nm, about 140 nm to about 150 nm, about 60 nm to about 130 nm, About 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm , 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm in average diameter, and is substantially non-toxic. The average diameter can be expressed as a z-average value determined by dynamic light scattering.

In one embodiment of the present disclosure, the zeta potential determined by dynamic light scattering is -50 to -150 mV, -50 to -140 mV, -50 to -130 mV, -50 to -120 mV, -50 to -110 mV, -50 to -100 mV, -50 to -90 mV, -50 to -80 mV, -60 to -150 mV, -60 to -140 mV, -60 to -130 mV, -60 to -120 mV , -60 to -110 mV, -60 to -100 mV, -60 to -90 mV, -60 to -80 mV, -70 to -150 mV, -70 to -140 mV, -70 to -130 mV, -70 to -120 mV, -70 to -110 mV, -70 to -100 mV, -70 to -90 mV, -70 to -80 mV, -75 to -150 mV, -75 to -140 mV, - 75 to -130 mV, -75 to -120 mV, -75 to -110 mV, -75 to -100 mV, -75 to -90 mV, or -75 to -80 mV, but is not limited thereto. .

In the present disclosure, mRNA encoding the S antigen of mutant SARS-CoV-2 or a portion thereof is adsorbed to the lipid portion of the liposome or lipid nanoparticle, or an aqueous solution surrounded by part or all of the lipid portion of the liposome or lipid nanoparticle. Encapsulated in space, mRNA or portions thereof may be protected from enzymatic degradation or other undesirable effects induced by mechanisms of the host organism or cell, such as negative immune responses.

The liposome or lipid nanoparticle may contain a cationic lipid.

In the present disclosure, 'cationic lipid' includes a lipid having cationic property continuously without the influence of pH change or an ionic lipid that is converted to cationic property by pH change.

The cationic lipids are 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), dimethyldioctadecylammonium bromide (DDA), 3β-[N-(N',N'-dimethylaminoethane carbamoyl Cholesterol (3β-[N-(N',N'-dimethylaminoethane) carbamoyl cholesterol, DC-Chol), 1,2-dioleoyloxy-3-dimethylammonium propane (DODAP), 1,2-di-O -Octadecenyl-3-triethylammonium propane (1,2-di-O-octadecenyl-3-trimethylammonium propane, DOTMA), 1,2-dimyristoleoyl-sn-glycero-3-ethylphos Forcolin (1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine, 14:1 Etyle PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (1-palmitoyl- 2-oleoyl-snglycero-3-ethylphosphocholine, 16:0-18:1 Ethyl PC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (1,2-dioleoyl-sn-glycero -3-ethylphosphocholine, 18:1 Ethyl PC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (1,2-distearoyl-sn-glycero-3-ethylphosphocholine, 18:0 Ethyl PC), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, 16:0 Ethyl PC), 1,2-di Myristoyl-sn-glycero-3-ethylphosphocholine (1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, 14:0 Ethyl PC), 1,2-dilauroyl-sn-glycero -3-Ethylphosphocholine (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 (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 (1,2-dimyristoyl-3-dimethylammonium-propane, 14:0 DAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (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 (N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1- aminium, DOBAQ), 1,2-stearoyl-3-trimethylammonium-propane (1,2-stearoyl-3-trimethylammoniumpropane, 18:0 TAP), 1,2-dipalmitoyl-3-trimethylammonium- Propane (1,2-dipalmitoyl-3-trimethylammonium-propane, 16:0 TA), 1,2-dimyristoyl-3-trimethylammonium-propane, 14:0 TAP) and/or N4-Cholesteryl-Spermine (GL67), preferably 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) or C12 -200, but is not limited thereto.

Specifically, the 'DOTAP (Dioleoyl-3-trimethylammonium propane)' is a cationic emulsifier having a structure of Chemical Formula 1, used as a fabric softener, and recently used as a nucleic acid carrier for forming liposomes.

[Formula 1]

The liposome or lipid nanoparticle may further include a neutral lipid.

In the present disclosure, 'neutral lipids' include lipids that have neutrality continuously without the influence of pH change or ionic lipids that are converted to neutrality by pH change.

The neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE), 1,2-dimyristoyl- sn-glycero-3-phosphatidylcholine (1,2-Dimyristoyl-sn-glycero-3-phosphorylcholine, DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (1,2-dioleoyl -sn-glycero-3-phosphocholine, DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC), 1, 2-distearoyl-sn-glycero-3-phosphocholine (1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), 1,2-dilinoleoyl-sn-glycero-3 -Phosphocholine (1,2-dilinoleoyl-sn-glycero-3-phosphocholine, DLPC), phosphatidylserine (PS), phosphoethanolamine (PE), phosphatidylglycerol (PG), phosphoric acid (PA) and / or may be phosphatidylcholine (PC), preferably 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE) It may, but is not limited thereto.

Specifically, 'DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine)' has a structure represented by Chemical Formula 2 below and is used as an auxiliary lipid for cationic liposomes.

[Formula 2]

In the present disclosure, the weight ratio of the cationic lipid to the neutral lipid is 1:9 to 9.5:0.5, preferably 2:8 to 9:1, more preferably 3:7 to 8:2, most preferably 4 :6 to 7:3, but is not limited thereto.

If the weight ratio is out of the range, mRNA delivery efficiency may be significantly reduced.

In addition, liposomes or lipid nanoparticles may additionally contain cholesterol.

In the present disclosure, the weight ratio of the cationic lipid to cholesterol is 6:1 to 1:3, preferably 4:1 to 1:2.5, more preferably 3:1 to 1:2, most preferably 2: It may be 1 to 1:1.5, but is not limited thereto.

In one embodiment of the present disclosure, the liposome or lipid nanoparticle may additionally include a PEG-modified lipid. The PEG modified lipid is PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkyl glycerol, or combinations thereof. In one embodiment, the PEG-modified lipid is, but is not limited to, DMG-PEG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG-DSG, PEG-DPG, or combinations thereof.

In addition, when the liposome or lipid nanoparticle according to the present disclosure contains both cationic lipid, neutral lipid and cholesterol, the weight ratio of the cationic lipid, neutral lipid and cholesterol is 1 to 9.5:9 to 0.5:0.05 to 3, Preferably 3 to 8:7 to 1:0.45 to 7.0, more preferably 1 to 3.5:1 to 3.5:0.5 to 3, but is not limited thereto.

If the weight ratio is out of the range, mRNA delivery efficiency may be significantly reduced.

Illustratively, when cholesterol is further included, liposomes may be prepared by mixing cholesterol at a weight ratio of 0.2 to 0.85, preferably 0.5 to 0.85, with respect to DOTAP:DOPE=1:1.

Furthermore, the liposome or lipid nanoparticle may additionally contain one or more delivery factors such as protamine, albumin, transferrin, protein transduction domains (PTD), cell penetrating peptide (CPP), and macrophage targeting moiety.

In the mutant SARS-CoV-2 vaccine composition of the present disclosure, the mixing ratio of liposomes or lipid nanoparticles and mRNA can be expressed as N:P ratio, and the N:P ratio affects the expression and stability of the delivery system .

The mutant SARS-CoV-2 vaccine composition of the present disclosure may additionally include an immune enhancer.

The immune enhancer corresponds to non-toxic lipooligosaccharide (dLOS), Pathogen-associated molecular pattern (PAMP) and responds to the pattern recognition receptor (PRR), CpG. It may be DNA, lipoprotein, flagella, poly I:C, saponin, squalene, tricaprin and/or 3D-MPL, but is not limited thereto.

Specifically, the non-toxic lipooligosaccharide (dLOS) may be a substance disclosed in Korean Patent Registration No. 1509456 or Korean Patent Registration No. 2042993, but is not limited thereto.

According to another aspect of the present disclosure, the present disclosure provides mRNA encoding the S antigen of the mutant SARS-CoV-2; And it provides a method for preparing a severe acute respiratory syndrome coronavirus 2 variant (SARS-CoV-2) vaccine composition comprising mixing liposomes or lipid nanoparticles.

The mRNA encoding the S antigen of the mutant SARS-CoV-2, and/or liposomes or lipid nanoparticles may be provided in the form of a lyophilized powder or dissolved in an appropriate solution or buffer. . When mRNA encoding the S antigen of mutant SARS-CoV-2 and/or liposome or lipid nanoparticles are provided in a lyophilized state, they can be used after being dissolved in an appropriate solution or buffer.

The method for preparing the mutant SARS-CoV-2 vaccine composition of the present disclosure may further include mixing an immune enhancer.

The adjuvant may be provided in the form of a lyophilized powder or dissolved in an appropriate solution or buffer. When the immunostimulant is provided in a lyophilized state, it may be used after being dissolved in an appropriate solution or buffer.

Since the mRNA encoding the S antigen of the mutant SARS-CoV-2 of the present disclosure and the liposome or lipid nanoparticle are active ingredients of the above-described mutant SARS-CoV-2 vaccine composition, the excessive complexity of the present specification is avoided for redundant information. To avoid that, the description is omitted.

According to another aspect of the present disclosure, the present disclosure provides a method for preventing a mutant SARS-CoV-2 infection comprising administering the above-described mutant SARS-CoV-2 vaccine to a subject.

Since the method for preventing mutant SARS-CoV-2 infection according to one aspect of the present disclosure includes the mutant SARS-CoV-2 vaccine composition according to one aspect of the present invention as a component, overlapping matters between the two inventions The same applies.

The route of administration of the mutant SARS-CoV-2 vaccine composition includes, but is not limited to, intradermal, intramuscular, subcutaneous, or intranasal administration. The preventive effect from the mutant SARS-CoV-2 virus due to administration of the vaccine composition may be obtained after at least 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after administration. there is.

The number of administrations may be once, twice, three times, four times or more, but there is a possibility that a sufficient preventive effect can be obtained with one administration.

In one embodiment of the present disclosure, the vaccine composition is formulated in an effective amount to generate an antigen-specific immune response (eg, production of antibodies specific to coronavirus antigens) in a subject. An "effective amount" is a dose of RNA effective to generate an antigen-specific immune response.

The immune response includes a humoral immune response and a cellular immune response.

A "humoral" immune response refers to an immune response mediated by antibody molecules, including, for example, secretory (IgA) or IgG molecules, whereas a "cellular" immune response refers to a T-lymphocyte (eg, It is an immune response mediated by CD4+ helper and/or CD8+ T cells (eg, CTL)) and/or other leukocytes.

The mutant SARS-CoV-2 vaccine according to the present disclosure exhibits excellent stability and high immunogenicity in vivo , making it easy to store and use the vaccine, and a SARS-CoV-2 mutant that has a low protective effect against infection with existing vaccines. Excellent preventive effect against viruses can be expected.

1 is a diagram showing the secondary structures of EG-COVARo mRNA, which is an mRNA of the present disclosure, and Omicron WT mRNA, which is a wild type.

Figure 2 is a diagram showing the results of measuring the IgG antibody titer when the EG-COVARo mRNA of the present disclosure was administered by dose.

3 is a diagram showing the ability to form neutralizing antibodies produced when EG-COVARo mRNA of the present disclosure is administered by dose.

4 is a diagram showing FRNT results to confirm the neutralizing ability of the antibody produced when the EG-COVARo mRNA of the present disclosure was administered by dose.

Example

Hereinafter, the present disclosure will be described in more detail through examples. These examples are only for explaining the present disclosure in more detail, and it will be apparent to those skilled in the art that the scope of the present disclosure is not to be construed as being limited by these examples.

ready yes. mRNA sequence selection

Sequence optimization was performed through the following method for the purpose of stabilizing mRNA by increasing the content of guanine and cytosine in mRNA and increasing translation efficiency in humans.

On the other hand, since the protein structure of the spike protein of SARS-CoV-2 is unstable, misfolding or triggering is prevented by proline substitution (2P) of amino acids 986(K) and 987(V), resulting in prefusion stabilized viral glycoprotein. It was confirmed through previous studies that it acts as a better immunogen.

Accordingly, in order to stabilize the protein structure of the spike protein, amino acids 986 (K) and 987 (V) were further mutated to proline, and a part of the amino acid sequence of 682 to 685 was additionally mutated, resulting in GenArt codon optimization program. The sequence of the mRNA was selected as the sequence. The obtained mRNA was named "EG-COVARo mRNA".

manufacturing example. Preparation of mRNA-liposome complex (mRNA-liposome)

1. mRNA synthesis

A plasmid containing a DNA sequence corresponding to the selected mRNA sequence was prepared by requesting Genscript (USA). The plasmid is pmRNA_T7_13_Kan_80A (2925 bp), consisting of T7, 5'-UTR, MCS, 3'-UTR, and poiy A tail (80) at the gene insertion part, and the SARS-CoV-2 antigen mutated at the MCS site A nucleotide having a nucleotide sequence encoding , for example, an mRNA sequence represented by the nucleotide sequence of SEQ ID NO: 1 is inserted and used. Using the plasmid, mRNA was obtained through an in vitro transcription (IVT) method. SARS-CoV-2 omicron mRNA was synthesized by replacing all uridines with 5-methoxyuridine (5moU, TriLink BioTechnologies) to reduce the antigenicity of the mRNA itself. 10X transcription buffer was prepared by mixing 1 M Tris-HCl (pH 8), 1 M Dithiothreitol (pH 8), 200 mM Spermidine, 10% Triton X-100, 1 M Magnesium acetate, and water for injection.

After mixing the 10X transcription buffer with 5moU, ATP, CTP, GTP, CleanCap AG, plasmid, RNase inhibitor, Pyrophosphate, and T7 Polymerase, they were reacted in a constant temperature water bath at 37°C.

DNase I (DNase I) and DNase I reaction buffer were added to the mixture and reacted in a constant temperature water bath at 37° C. to remove DNA.

Water for injection, 10% SDS solution, 1 M Dithiothreitol (pH 8), and Proteinase K were added to the mixture and reacted in a constant temperature water bath at 37° C. to remove proteins.

The mRNA was purified through Cation-exchange-HPLC and a diafiltration system, the buffer was replaced, and the mRNA was obtained by sterilization filtration.

2. Liposome preparation (Film method)

DOTAP (Merck & Cie/CH2900014), DOPE (Avanti Polar Lipid), and/or cholesterol (Avanti Polar Lipid) are mixed with chloroform, respectively, to a concentration of 3 mg/mL, and completely dissolved at 37 °C for 10 minutes to prepare a liquid solution. did

A lipid mixture was prepared by mixing the liquid solution at a certain weight ratio in a round bottom flask, and volatilized at 60 ° C. for 30 minutes in a rotary evaporator (Buchi / B491_R200) to blow chloroform to form a lipid film on the flask wall.

Add 20 mM HEPES buffer (pH 7.4) containing 4% (w/v) sucrose to the flask in which the lipid film film was prepared, melt the lipid film at 60 ° C, and prepare liposomes to a liposome concentration of 7.5 mg / mL. formed. The formed liposomes were measured for particle size, zeta potential, and dispersion using dynamic light scattering analysis equipment. The remaining prepared liposomes were stored at 4°C until testing.

3. Preparation of mRNA-liposome complexes

Mix the prepared liposome (LP-DOTAP/DOPE/Chol (40:40:20, w/w/w)) and mRNA in 20 mM HEPES buffer (pH 7.4) containing 4% sucrose at room temperature. Thus, an mRNA-liposome complex (100 μl) was prepared. The mixing ratio of liposome and mRNA, N/P ratio, was calculated by the following formula.

[Equation 1]

The composites prepared are as follows:

- Omicron-PQ SARS-CoV-2 S protein mRNA (SEQ ID NO: 1)-liposome complex

Example 1. mRNA secondary structure analysis

The secondary structure of EG-COVARo mRNA was analyzed using the loop-based energy model and dynamic programming algorithm introduced by Zuker et al.

The results are shown in Figure 1.

As shown in FIG. 1, when comparing Omicron WT mRNA and EG-COVARo mRNA (codon optimized) minimum free energy (MFE), EG-COVARo of the present disclosure is -1344.50 kcal/mol, which is much more thermodynamic than WT of -1067.60 kcal/mol. was stable. In addition, in the structural image analysis results, EG-COVARo of the present disclosure had higher distributions of red, yellow, and green, and thus had higher base pair probabilities than WT, which had higher distributions of blue and green. Thus, it was shown that EG-COVARo of the present disclosure retains a stable secondary structure better than WT.

Example 2. DLS (Dynamic Light Scattering) analysis of mRNA-liposome complexes

The mRNA-liposome complex prepared in Preparation Example was diluted 1/10 with 20 mM HEPES buffer (pH 7.4) containing 4% sucrose. DLS analysis was performed with a Zetasizer Nano ZSP (Malvern Pnanlytical) to measure the size, polydispersity index (PDI), and zeta potential of the complex.

The results are shown in Table 1 below.

Complex size (d. nm)	dispersion	zeta potential
149.45±4.15	0.1175±0.082	-77.60±0.53

As shown in Table 1, the mRNA-liposome complex of the present disclosure showed that particles of about 149 nm in size were relatively evenly distributed, and the degree of dispersion was close to 0.1 and the zeta potential was low, so it was dispersed without aggregation and had high structural stability. .

Example 3. Confirmation of immunogenicity of mRNA-liposome complex (confirmation of IgG antibody titer)

With respect to the liposome-mRNA complex prepared in the above Preparation Example, the following experiment was conducted. In each experiment, liposome-mRNA complexes prepared to contain different doses of mRNA were administered to 6-week-old female mice (B6C3F1/slc, central experimental animal) by intramuscular administration to the left hind thigh of the mouse. Table 2 shows the substance to be administered, the administration method, and the administration dose for each specific group.

serial number	Group	Dosing Details (per mouse)	dosing regimen	dose
One	negative control group	4% sucrose in 20 mM HEPES buffer (pH 7.4)	2 times every 3 weeks intramuscular administration	100 µL
2	EG-COVARo 5 μg	5 μg mRNA + Liposome
3	EG-COVARo 10 μg	10 μg mRNA + Liposome
4	EG-COVARo 20 μg	20 μg mRNA + Liposome

Two weeks after the last administration, Avertin working solution was administered intraperitoneally at 250 mg/kg to anesthetize, and whole blood was collected through cardiac blood sampling. The collected whole blood was transferred to a microtube, allowed to stand at room temperature for 3 hours, centrifuged at 4°C and 15,000 rpm for 10 minutes, and the supernatant was transferred to a new microtube to obtain serum and stored at -20°C until analysis. did

Next, after diluting the SARS-CoV-2 Omicron RBD (SARS-CoV-2 receptor binding domain) antigen (Mybiosource, USA) to 1 μg/mL using 1X PBS, 100 μl/ml was added to the immunoplate. The wells were dispensed, covered with a sealing film, and allowed to stand overnight at 4°C. The solution in each well was removed with an ELISA washer (Tecan/Hydroflexelisa) and washed three times using a washing buffer (500 μl of tween 20 was added to 1 L of 1X PBS in which 20X PBS was diluted with purified water). Reagent diluent (reagent diluent, 1% BSA, prepared by dissolving 1 g of BSA in 100 mL of PBS) was dispensed into the immunoplate at a rate of 200 μl/well, covered with a sealing film, and allowed to stand in a 37° C. reactor for 1 hour. The solution in each well was removed with an ELISA washer and washed three times using a washing buffer. The reagent diluent was dispensed into the immunoplate at 100 μl/well.

After diluting the serum obtained above 1:50 using a reagent diluent (1% BSA), dispense 100 μl in rows B to F of the immunoplate, and mix the samples by pipetting several times in the well After that, the sample was serially diluted 1/2 up to column 12 on the ELISA plate by taking 100 μl from column 1 and putting it in column 2. At this time, in order to confirm the equivalence of the experimental conditions between the plates, hyperserum was diluted 1:100 using a reagent diluent, and then 100 μl was dispensed in column 1 of each immunoplate H, and 1 /2 serial dilution.

The immunoplate was covered with a sealing film and reacted in a 37° C. reactor for 2 hours. The solution in each well was removed with an ELISA washer and washed 5 times using a wash buffer. After diluting goat anti-mouse IgG antibody (Jackson Laboratory) 1:5,000 using a reagent diluent, 100 μl of each was dispensed into an immunoplate, covered with a sealing film, and reacted for 1 hour in a 37 °C reactor. The solution in each well was removed with an ELISA washer and washed three times using a washing buffer.

100 μl/well of the TMB substrate solution equilibrated with room temperature was dispensed into the immunoplate and reacted for 3 minutes and 30 seconds in the dark at room temperature. The reaction was stopped by dispensing 100 μl of the 1N H ₂ SO ₄ solution into the immunoplate, and the absorbance was measured at 450 nm using an ELISA reader (TECAN/SPARK).

The results are shown in Figure 2.

As shown in FIG. 2, as a result of the analysis, the antibody titer was significantly increased compared to the negative control group in the EG-COVARo administration group at all doses (p<0.01). In particular, in the 5 μg/head and 10 μg/head administration groups, an increase in antibody titer was observed with increasing dose.

Example 4. Neutralization (%)

SARS-CoV-2 surrogate virus neutralization test (sVNT) kit (Genscript) was used for serum samples obtained by administering the EG-COVARo vaccine of the present disclosure to confirm whether the vaccine effectively inhibits viral infection.

After dispensing 63 μl of sample dilution buffer to tubes 1 to 5 of the 8-strip tube, put 7 μl of serum sample in tube 1, pipette several times in the tube, take 7 μl, and put it into the next tube. Samples were serially diluted to the fifth tube to obtain diluted serum samples.

In a 1.5mL microtube, 6μl of the negative control and positive control were mixed with 54μl of sample dilution buffer and diluted 1:10, and the diluted serum sample was dispensed to the immunoplate at 60μl/well, followed by 1:1000 diluted-HPR. After adding 60 μl of conjugated Omicron RBD, the mixture was reacted at 37° C. for 30 minutes. 100 μl of the mixture was dispensed on a microtiter test strip plate, covered with a sealing film, and reacted at 37° C. for 15 minutes.

The solution in each well was removed and washed 4 times with 1X washing solution. After dispensing 100 μl/well of the TMB solution and reacting for 15 minutes in the dark at room temperature by covering the sealing film, stop the reaction by dispensing the stop solution by 50 μl/well, and then using an ELISA reader (TECAN/SPARK) at 450 nm After measuring the optical density, Omicron RBD protein neutralizing ability (sVNT, %) was calculated by the following formula.

[Equation 2]

The value (Inhibitory Concentration 50 titer, IC ₅₀ titer) at which Omicron RBD protein neutralizing ability (%) is reduced by 50% is Nonlinear regression of GraphPad Prism software → dose-response (Inhibition) → log (inhibitor) vs. It was derived through the normalized response.

The results are shown in Figure 3.

As shown in FIG. 3, in the 5 μg/head and 10 μg/head administration groups, a tendency for the neutralizing ability to Omicron RBD protein to increase with increasing dose was observed.

Example 5. Focus Reduction Neutralization test (FRNT) analysis

In order to confirm the neutralizing ability of the SARS-CoV-2 Omicron variant virus of the present disclosure, a Focus Reduction Neutralization test (FRNT) was performed using the serum collected in Example 3.

Vero cells were seeded in a 96-well plate, and the next day, serum was first diluted 1:20 and serially diluted 4-fold. After mixing the diluted serum and Omicron virus (NCCP43408) at a ratio of 1:1 (v/v) and incubating for 30 minutes, the Vero cells were washed and infected with the serum-Omicron virus mixture.

After 8 hours of infection, the infected Vero cells were fixed with 10% formalin, labeled with anti-SARS-CoV-2 NP antibody, and then colored with TrueBlue, and the number of foci was measured using an ELISPOT reader.

The results are shown in FIG. 4 .

As shown in FIG. 4, the neutralizing antibody titer significantly increased compared to the negative control group in the EG-COVARo administered group at all doses. (p<0.01) In particular, it was confirmed that the neutralizing antibody titer increased depending on the administered dose.

Since certain parts of the present disclosure have been described in detail above, it is clear that these specific descriptions are only preferred implementation examples for those skilled in the art, and the scope of the present disclosure is not limited thereto.

Claims (13)

1. A mutant SARS-CoV-2 vaccine composition comprising mRNA encoding a Spike antigen of severe acute respiratory syndrome coronavirus 2 variant (SARS-CoV-2).
2. The mutant SARS-CoV-2 vaccine composition according to claim 1, wherein the mutation is an Omicron mutation.
3. The mutant SARS-CoV-2 vaccine composition according to claim 1, wherein the mRNA has the nucleotide sequence of SEQ ID NO: 1.
4. The mutant SARS-CoV-2 vaccine composition according to claim 1, wherein the Spike antigen has the amino acid sequence of SEQ ID NO: 2.
5. The mutant SARS-CoV-2 vaccine composition according to claim 1, wherein the composition further comprises liposomes or lipid nanoparticles.
6. The mutant SARS-CoV-2 vaccine composition according to claim 5, wherein the liposome or lipid nanoparticle comprises a cationic lipid.
7. The mutant SARS-CoV-2 vaccine composition according to claim 6, wherein the liposome or lipid nanoparticle further comprises a neutral lipid.
8. The mutant SARS-CoV-2 vaccine composition according to claim 6, wherein the liposome or lipid nanoparticle further comprises cholesterol.
9. The mutant SARS-CoV-2 vaccine composition according to claim 7, wherein the weight ratio of the cationic lipid and the neutral lipid is 1:9 to 9.5:0.5.
10. According to claim 8, wherein the weight ratio of the cationic lipid and cholesterol is 6: 1 to 1: 3, the mutant SARS-CoV-2 vaccine composition.
11. 11. The method of claim 10, wherein the weight ratio of the cationic lipid, neutral lipid and cholesterol is 1 to 9.5: 9 to 0.5: 0.05 to 3, variant SARS-CoV-2 vaccine composition.
12. The mutant SARS-CoV-2 vaccine composition according to claim 4, wherein the liposome or lipid nanoparticle and mRNA have an N:P ratio of 0.23:1 to 1.39:1.
13. The mutant SARS-CoV-2 vaccine composition according to any one of claims 1 to 12, wherein the composition further comprises an immunostimulant.

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