Alphavirus-based vector system for producing virus-like particle comprising self-amplifying RNA

Abstract

One aspect of the present invention provides: a vector including a gene encoding a non-structural alphavirus protein of and a gene encoding a target protein; a cell infected with the vector; a virus-like particle (VLP); and a vaccine composition comprising same. The vector is a single-vector system and can easily and conveniently produce VLP containing saRNA without a helper vector. The VLP and vaccine composition produced through the vector produce a sufficient amount of antibody from only a single administration, have excellent safety due to the absence of additional proliferation, can be administered to mucous membranes, and induces mucous membrane immunity, and thus exhibits a remarkably superior respiratory disease prevention or treatment effect compared to therapeutic agents for intramuscular administration.

Description

Alphavirus-based vector system for the production of virus-like particles containing self-amplifying RNA

[0001] It relates to a vector comprising a gene encoding a nonstructural protein of an alphavirus and a gene encoding a target protein.

[0002]

[0003] A vaccine is a medicinal product containing a weakened pathogen or a protein or nucleic acid that has been processed to allow the human body to recognize its information. Depending on the manufacturing method, vaccines can be broadly categorized as live attenuated vaccines, inactivated vaccines, toxoid vaccines, mRNA vaccines, extracted vaccines, viral vector vaccines, and virus-like particle (VLP) vaccines.

[0004] People are exposed to external pathogens through the respiratory system, most of which invade through the mucous membranes. Mucosal immunity, which serves as the first line of defense against these pathogens, is the most effective means of preventing pathogen invasion.

[0005] Mucosal vaccines can induce not only a mucosal immune response but also a systemic immune response. They can produce antigen-specific IgA on the mucosal surface, a key feature of mucosal immunity. Furthermore, they offer the advantage of being safe and easy to administer, as they do not require needles for injection, causing no pain or discomfort. Currently, mucosal vaccines composed of attenuated live or killed vaccines are commercially available. However, live vaccines have raised safety concerns, and killed vaccines, while safer than live vaccines, have the disadvantage of having low immunogenicity and making it difficult to induce an effective immune response. Therefore, there is a pressing need to address issues related to the efficacy and safety of these mucosal vaccines.

[0006] mRNA vaccines induce an immune response by administering mRNA containing the virus's genetic information, causing the body to produce antigens directly. However, mRNA vaccines contain only the sequence encoding the antigen protein, which means they do not produce enough antigen to induce immunity, requiring a high dose. Furthermore, a stable carrier capable of delivering the mRNA into the body is required.

[0007] VLP (Virus-Like Particle) vaccines are particles that specifically express the structural proteins of a virus to have a structure similar to that of a wild-type virus. Since VLP vaccines do not contain genetic information about the pathogen inside the particle, they can be used as a vector, and since they have a structure similar to the appearance of the virus, they can be administered through the infection route of existing viruses to induce an immune response.

[0008] Accordingly, the present inventors developed a vector system for producing a VLP vaccine containing self-amplifying saRNA (self-amplifying RNA), and the VLP vaccine produced through the vector system can be administered to the mucosa, enabling mucosal immunity and systemic immunity therethrough, thereby revealing remarkably excellent efficacy as a mucosal immunity vaccine for the prevention or treatment of respiratory diseases.

[0009]

[0010] One aspect is to provide a vector comprising a gene encoding a nonstructural protein of an alphavirus and a gene encoding a protein of interest.

[0011] Another aspect provides a method for producing cells for producing virus like particles (VLPs), comprising the step of introducing RNA expressed by the vector into the cells.

[0012] Another aspect provides a cell for producing virus-like particles (VLPs) manufactured by the method for producing the cell for producing virus-like particles (VLPs).

[0013] Another aspect provides a method for producing virus-like particles, comprising a step of culturing cells for producing the virus-like particles.

[0014] Another aspect is to provide a virus-like particle manufactured by the method for manufacturing the virus-like particle.

[0015] Another aspect is to provide a vaccine composition for preventing or treating respiratory diseases comprising the virus-like particles.

[0016] Another aspect provides a method for preventing or treating a respiratory disease, comprising administering to a subject in need thereof a vaccine composition comprising the virus-like particles.

[0017] Another aspect provides the use of the virus-like particles for the manufacture of a medicament for preventing or treating the above respiratory disease.

[0018]

[0019] One aspect provides a vector comprising a gene encoding a nonstructural protein of an alphavirus and a gene encoding a protein of interest.

[0020] The term "alphavirus" refers to a virus belonging to the Togaviridae family, a small, spherical, enveloped virus with a single positive-sense strand RNA genome. It can infect a wide range of cells (including insects, birds, and mammals), and its RNA genome itself encodes an RNA replicase, allowing efficient RNA replication and transcription. The alphavirus genome contains two open reading frames (ORFs): a nonstructural and a structural one. The first ORF contains the NSP genes, which encode proteins essential for the transcription and translation of viral RNA (NSP1 to NSP4), producing a single polyprotein precursor that is proteolytically cleaved into the mature protein. The second ORF encodes three structural proteins: the core nucleocapsid protein C, the envelope protein P62, and E1.

[0021] The term "protein" refers to a molecule composed of amino acids linked by peptide bonds, and may be used interchangeably with polypeptide. Such proteins include recombinant proteins, portions of proteins, or fragments thereof. For example, they may include chimeric proteins, peptides or polypeptides of a size that can function as epitopes, and the like.

[0022] The term "gene" should be considered in the broadest sense and may be DNA or RNA (specifically mRNA), and for example, if the gene is DNA, it may be in a form operable and contained in a vector.

[0023] The term "coding gene" refers to a gene having a base sequence that encodes the amino acid sequence of a polypeptide or protein. The "coding gene" includes a gene that encodes a portion of the polypeptide or a fragment thereof, or a gene that encodes a portion of the protein or a fragment thereof.

[0024] The term "vector" refers to a nucleic acid molecule used to transport genetic material to another cell, where it can be replicated and / or expressed. Examples include plasmids, viral vectors (bacteriophages, animal viruses, and plant viruses), cosmids, and artificial chromosomes (e.g., YACs). Specifically, the vector may be a plasmid, and more specifically, a plasmid derived from an alpha virus, such as a plasmid derived from the Semliki Forest Virus.

[0025] In one specific example, the alpha virus is Aura virus, Babanki virus, Barmah Forest virus, Bebaru virus, Cabassou virus, Chikungunya virus, Eastern equine encephalitis virus, Everglades virus, Fort Morgan virus, Getah virus, Highlands J virus, Kyzylagach virus, Mayaro virus, Me Tri virus, Middelburg virus, Mosso das Pedras virus, Mucambo virus, Ndumu virus, O'nyong-nyong virus, Pixuna The virus may be at least one selected from the group consisting of Rio Negro virus, Ross River virus, Salmon pancreas disease virus, Semliki Forest virus (SFV), Southern elephant seal virus, Tonate virus, Trocara virus, Una virus, Western equine encephalitis virus and Whataroa virus, and specifically may be Semliki Forest virus (SFV).

[0026] For example, the nonstructural protein of the alphavirus may be a nonstructural protein derived from one or more alphaviruses. For example, the nonstructural protein of the alphavirus may be a combination of nonstructural proteins derived from two different alphaviruses (a nonstructural protein derived from Aura virus and a nonstructural protein derived from Semliki Forest virus). Furthermore, the nonstructural proteins of the alphavirus may be nonstructural proteins derived from the same alphavirus. For example, the nonstructural protein of the alphavirus may be a nonstructural protein derived from Semliki Forest virus.

[0027] In one specific example, the nonstructural protein of the alphavirus may be at least one selected from the group consisting of NSP1 protein, NSP2 protein, NSP3 protein, and NSP4 protein, and specifically may include all of the NSP1 protein, NSP2 protein, NSP3 protein, and NSP4 protein of the alphavirus. More specifically, it may include all of the NSP1 protein, NSP2 protein, NSP3 protein, and NSP4 protein of the Semliki Forest Virus.

[0028] The NSP1 protein has methyltransferase activity and may be involved in the viral capping reaction. The NSP2 protein may have helicase and protease activities. The NSP3 protein comprises three domains: a macrodomain, a central (or alphavirus-unique) domain, and a hypervariable domain (HVD). The NSP4 protein has RNA-dependent RNA polymerase activity. That is, it may comprise part or all of an RNA-dependent RNA polymerase, and may comprise an element (e.g., a protein, an enzyme, etc.) capable of activating an RNA-dependent RNA polymerase.

[0029] In one specific example, the nonstructural protein of the alpha virus may include an RNA-dependent RNA polymerase (RdRp). Specifically, it may include an RNA-dependent RNA polymerase (RdRp) of the Semliki Forest Virus.

[0030] The above RNA-dependent RNA polymerase is an enzyme that replicates RNA using RNA as a template, and is found in archaea and some viruses.

[0031] In one specific example, the gene encoding a nonstructural protein of an alphavirus may include one or more genes selected from the group consisting of a gene encoding an NSP1 protein of the alphavirus, a gene encoding an NSP2 protein of the alphavirus, a gene encoding an NSP3 protein of the alphavirus, and a gene encoding an NSP4 protein of the alphavirus, and specifically, may include a gene encoding an NSP1 protein of the alphavirus, a gene encoding an NSP2 protein of the alphavirus, a gene encoding an NSP3 protein of the alphavirus, and a gene encoding an NSP4 protein of the alphavirus. More specifically, may include a gene encoding an NSP1 protein of a Semliki forest virus, a gene encoding an NSP2 protein of a Semliki forest virus, a gene encoding an NSP3 protein of a Semliki forest virus, and a gene encoding an NSP4 protein of a Semliki forest virus.

[0032] In one specific example, the gene encoding the nonstructural protein of the alpha virus may include a gene encoding an RNA-dependent RNA polymerase (RdRp), and specifically, the gene encoding the NSP1 protein of the alpha virus, the gene encoding the NSP2 protein of the alpha virus, the gene encoding the NSP3 protein of the alpha virus, and the gene encoding the NSP4 protein of the alpha virus may include a gene encoding an RNA-dependent RNA polymerase (RdRp), and more specifically, the gene encoding the NSP1 protein of the Semliki Forest Virus, the gene encoding the NSP2 protein of the Semliki Forest Virus, the gene encoding the NSP3 protein of the Semliki Forest Virus, and the gene encoding the NSP4 protein of the Semliki Forest Virus may include a gene encoding an RNA-dependent RNA polymerase (RdRp).

[0033] In addition, in one specific example, the vector may include a gene encoding an RNA-dependent RNA polymerase of the alphavirus, and specifically, a gene encoding an RNA-dependent RNA polymerase of the alphavirus and a gene encoding an NSP1 protein of the alphavirus, a gene encoding an NSP2 protein of the alphavirus, a gene encoding an NSP3 protein of the alphavirus, and a gene encoding an NSP4 protein of the alphavirus. More specifically, the vector may include a gene encoding an RNA-dependent RNA polymerase of the Semliki forest virus and a gene encoding an NSP1 protein of the Semliki forest virus, a gene encoding an NSP2 protein of the Semliki forest virus, a gene encoding an NSP3 protein of the Semliki forest virus, and a gene encoding an NSP4 protein of the Semliki forest virus.

[0034] Specifically, when the vector includes a gene encoding NSP1 to NSP4 proteins among the nonstructural proteins of the alpha virus, when the vector is expressed, all or part of the RNA-dependent RNA polymerase or an element capable of activating the RNA-dependent RNA polymerase is also expressed, so that the RNA-dependent RNA polymerase can function normally.

[0035] In one specific example, the gene encoding the NSP1 protein of the alpha virus may be a polynucleotide consisting of a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 1, and specifically, the gene encoding the NSP1 protein of the alpha virus may be a polynucleotide consisting of a base sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, or 100% homology with the base sequence of SEQ ID NO: 1.

[0036] In one specific example, the gene encoding the NSP2 protein of the alpha virus may be a polynucleotide consisting of a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 3, and specifically, the gene encoding the NSP2 protein of the alpha virus may be a polynucleotide consisting of a base sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or 100% homology with the base sequence of SEQ ID NO: 3.

[0037] In one specific example, the gene encoding the NSP3 protein of the alpha virus may be a polynucleotide consisting of a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 5, and specifically, the gene encoding the NSP3 protein of the alpha virus may be a polynucleotide consisting of a base sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or 100% homology with the base sequence of SEQ ID NO: 5.

[0038] In one specific example, the gene encoding the NSP4 protein of the alpha virus may be a polynucleotide consisting of a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 7, and specifically, the gene encoding the NSP4 protein of the alpha virus may be a polynucleotide consisting of a base sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, or 100% homology with the base sequence of SEQ ID NO: 7.

[0039] In one specific example, the gene encoding the RNA-dependent RNA polymerase of the alpha virus may be a polynucleotide consisting of a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 9, and specifically, the gene encoding the RNA-dependent RNA polymerase of the alpha virus may be a polynucleotide consisting of a base sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more or 100% homology with the base sequence of SEQ ID NO: 9. It may be a polynucleotide consisting of a sequence.

[0040] In one specific example, the vector may further comprise a regulatory sequence.

[0041] The above "regulatory sequence" means a nucleic acid having an expression activity that regulates the expression of the nucleic acid or the gene, i.e., transcription and translation, after being functionally linked to the expression target sequence or gene, and may include a promoter, enhancer, and other expression regulatory elements (e.g., polyadenylation signals and elements affecting the stability of expressed RNA).

[0042] In one specific example, the regulatory sequence may include at least one selected from the group consisting of a 3'-untranslated region (UTR) sequence, a 5'-UTR sequence, and a poly(A) tail sequence, and specifically may include all of a 3'-UTR sequence, a 5'-UTR sequence, and a poly(A) tail sequence.

[0043] The term "3' untranslated region (3'-UTR)" refers to a region located at the 3' end of the open reading frame that plays a role in controlling gene expression.

[0044] The term "5' untranslated region (5'-UTR)" refers to a region located at the 5' end of the open reading frame and is involved in the initiation of protein synthesis.

[0045] The above term "poly(A) tail" refers to a structure located at the 3' terminal region that affects expression efficiency by delaying the degradation process of exoribonuclease and extending the stability and in vivo half-life of expressed RNA, and can generally be formed by a plurality of adenine nucleotide sequences.

[0046] In one specific example, the regulatory sequence may be operably linked.

[0047] The term "operably linked" means that the introduced gene is connected to regulatory sequences in such a way that it can be expressed in the host cell.

[0048] The term "respiratory disease-causing virus" refers to a virus that causes respiratory disease by infecting mucous membranes, such as the upper respiratory tract (upper respiratory tract), lower respiratory tract (lower respiratory tract), eye mucosa, nasal mucosa, or oral mucosa, through droplet transmission or direct contact transmission.

[0049] In one specific example, the target protein may be a target virus-derived protein or a fragment thereof. Specifically, the target protein may be a target virus-derived protein or a fragment thereof, and the target virus-derived protein or a fragment thereof may be at least one selected from the group consisting of a target virus-derived spike protein or a fragment thereof, a target virus-derived envelope protein or a fragment thereof, a target virus-derived membrane protein or a fragment thereof, a target virus-derived packaging signal-related protein or a fragment thereof, and a target virus-derived nucleocapsid protein or a fragment thereof. More specifically, the target virus-derived protein or a fragment thereof may be a target virus-derived spike protein or a fragment thereof, a target virus-derived envelope protein or a fragment thereof, a target virus-derived membrane protein or a fragment thereof, a target virus-derived packaging signal-related protein or a fragment thereof, and a target virus-derived nucleocapsid protein or a fragment thereof; Alternatively, the target virus-derived protein or fragment thereof may be a target virus-derived spike protein or fragment thereof, a target virus-derived envelope protein or fragment thereof, a target virus-derived membrane protein or fragment thereof, and a target virus-derived packaging signal-related protein or fragment thereof.

[0050] The term "Spike (S) protein" refers to a structural protein present on the surface of the virus, consisting of club-shaped protrusions. The spike protein is known to bind to a host cell's glycoprotein receptor, which can lead to fusion of the cell membrane with the viral outer membrane and the production of neutralizing antibodies.

[0051] The term "Envelope (E) protein" constitutes the outermost layer of the virus and serves to protect the viral genome.

[0052] The term "membrane (M) protein" refers to a transmembrane protein that plays a central role in the assembly of the virus particle.

[0053] The term "nucleocapsid (N) protein" plays an important role in binding to viral RNA and stabilizing and packaging the genome during the assembly, envelope formation, and RNA synthesis of viral particles.

[0054] The term "packaging signal" is found in the positive-sense single-stranded RNA genome, and this packaging signal enables the structural proteins of the virus to assemble into a viral particle. The term "packaging signal-associated protein" refers to a protein that plays a role in inducing particle assembly, such as a nonstructural protein, enzyme, or small molecule protein.

[0055] In one specific example, the gene encoding the target protein may be a gene encoding a target virus-derived protein or a fragment thereof. Specifically, the gene encoding the target protein may be a gene encoding a target virus-derived protein or a fragment thereof, and the gene encoding the target virus-derived protein or a fragment thereof may be at least one selected from the group consisting of a gene encoding a target virus-derived spike protein or a fragment thereof, a gene encoding a target virus-derived envelope protein or a fragment thereof, a gene encoding a target virus-derived membrane protein or a fragment thereof, a gene encoding a target virus-derived packaging signal-related protein or a fragment thereof, and a gene encoding a target virus-derived nucleocapsid protein or a fragment thereof. More specifically, it may be a gene encoding a target virus-derived spike protein or a fragment thereof, a gene encoding a target virus-derived envelope protein or a fragment thereof, a gene encoding a target virus-derived membrane protein or a fragment thereof, a gene encoding a target virus-derived packaging signal-related protein or a fragment thereof, and a gene encoding a target virus-derived nucleocapsid protein or a fragment thereof; or the gene encoding the target virus-derived protein or a fragment thereof may be a gene encoding a target virus-derived spike protein or a fragment thereof, a gene encoding a target virus-derived envelope protein or a fragment thereof, a gene encoding a target virus-derived membrane protein or a fragment thereof, and a gene encoding a target virus-derived packaging signal-related protein or a fragment thereof.

[0056] In one specific example, the target virus may be a respiratory disease-causing virus. Specifically, the target virus-derived protein or a fragment thereof may be a respiratory disease-causing virus-derived protein or a fragment thereof. More specifically, it may be at least one selected from the group consisting of a respiratory disease-causing virus-derived spike protein or a fragment thereof, a respiratory disease-causing virus-derived envelope protein or a fragment thereof, a respiratory disease-causing virus-derived membrane protein or a fragment thereof, a respiratory disease-causing virus-derived packaging signal-related protein or a fragment thereof, and a respiratory disease-causing virus-derived nucleocapsid protein or a fragment thereof. Even more specifically, the target virus-derived protein or a fragment thereof may be a respiratory disease-causing virus-derived spike protein or a fragment thereof, a respiratory disease-causing virus-derived envelope protein or a fragment thereof, a respiratory disease-causing virus-derived membrane protein or a fragment thereof, a respiratory disease-causing virus-derived packaging signal-related protein or a fragment thereof, and a respiratory disease-causing virus-derived nucleocapsid protein or a fragment thereof; Alternatively, the target virus-derived protein or fragment thereof may be a respiratory disease-causing virus-derived spike protein or fragment thereof, a respiratory disease-causing virus-derived envelope protein or fragment thereof, a respiratory disease-causing virus-derived membrane protein or fragment thereof, and a respiratory disease-causing virus-derived packaging signal-related protein or fragment thereof.

[0057] For example, if the target virus is a coronavirus, and the target virus-derived protein or a fragment thereof is a coronavirus spike protein or a fragment thereof, the target virus-derived protein or a fragment thereof means the whole, a part thereof, or a fragment thereof of the coronavirus spike protein, and specifically, means the whole or a part thereof of the coronavirus spike protein or a fragment thereof of the coronavirus spike protein or a part thereof.

[0058] In addition, the target virus-derived protein or fragment thereof may be produced by recombining a protein or fragment thereof derived from a different mutant virus of the same lineage or a virus of a different lineage. For example, if the target virus-derived protein or fragment thereof is the spike protein of the SARS-CoV-2 virus or a fragment thereof, it may refer to a chimeric spike protein or fragment thereof in which the receptor binding domain (RBD) portion of the spike protein of the SARS-CoV-2 virus or a fragment thereof is recombined with the receptor binding domain of the MERS-CoV virus.

[0059] In one specific example, the gene encoding the target virus-derived protein or a fragment thereof may be a gene encoding a respiratory disease-causing virus-derived protein or a fragment thereof. Specifically, it may be at least one selected from the group consisting of a gene encoding a respiratory disease-causing virus-derived spike protein or a fragment thereof, a gene encoding a respiratory disease-causing virus-derived envelope protein or a fragment thereof, a gene encoding a respiratory disease-causing virus-derived membrane protein or a fragment thereof, a gene encoding a respiratory disease-causing virus-derived packaging signal-related protein or a fragment thereof, and a gene encoding a respiratory disease-causing virus-derived nucleocapsid protein or a fragment thereof, and more specifically, it may be a gene encoding a respiratory disease-causing virus-derived spike protein or a fragment thereof, a gene encoding a respiratory disease-causing virus-derived envelope protein or a fragment thereof, a gene encoding a respiratory disease-causing virus-derived membrane protein or a fragment thereof, a gene encoding a respiratory disease-causing virus-derived packaging signal-related protein or a fragment thereof, and a gene encoding a respiratory disease-causing virus-derived nucleocapsid protein or a fragment thereof; Alternatively, the gene encoding the target virus-derived protein or a fragment thereof may be a gene encoding a respiratory disease-causing virus-derived spike protein or a fragment thereof, a gene encoding a respiratory disease-causing virus-derived envelope protein or a fragment thereof, a gene encoding a respiratory disease-causing virus-derived membrane protein or a fragment thereof, and a gene encoding a respiratory disease-causing virus-derived packaging signal-related protein or a fragment thereof.

[0060] For example, if the target virus is a coronavirus, and the target virus-derived protein or a fragment thereof is a coronavirus spike protein or a fragment thereof, the gene encoding the target virus-derived protein or a fragment thereof means a gene encoding the whole, a part thereof, or a fragment thereof of the coronavirus spike protein, and specifically, means a gene encoding the whole or a part thereof of the coronavirus spike protein, or a gene encoding a fragment or a part thereof of the coronavirus spike protein.

[0061] In addition, the gene encoding the target virus-derived protein or a fragment thereof may be a gene encoding a protein produced by recombining a protein or a fragment thereof derived from a different mutant virus of the same lineage or a virus of a different lineage. For example, if the gene encoding the target virus-derived protein or a fragment thereof is a gene encoding the spike protein of the SARS-CoV-2 virus or a fragment thereof, it may refer to a gene encoding a chimeric spike protein or a fragment thereof, in which the receptor binding domain (RBD) portion of the spike protein of the SARS-CoV-2 virus or a fragment thereof is recombined with the receptor binding domain of the MERS-CoV virus.

[0062] In one specific example, the gene encoding the target virus-derived spike protein may be a polynucleotide consisting of a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 11, SEQ ID NO: 23 or SEQ ID NO: 25, and specifically, the gene encoding the target virus-derived spike protein may be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more homology with the base sequence of SEQ ID NO: 11, SEQ ID NO: 23 or SEQ ID NO: 25, or It may be a polynucleotide consisting of a base sequence with 100% homology.

[0063] In one specific example, the gene encoding the coat protein may be a polynucleotide consisting of a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 13, and specifically, the gene encoding the coat protein may be a polynucleotide consisting of a base sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more or 100% homology with the base sequence of SEQ ID NO: 13.

[0064] In one specific example, the gene encoding the membrane protein may be a polynucleotide consisting of a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 15, and specifically, the gene encoding the membrane protein may be a polynucleotide consisting of a base sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more or 100% homology with the base sequence of SEQ ID NO: 15.

[0065] In one specific example, the gene encoding the nucleocapsid protein may be a polynucleotide consisting of a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 17, and specifically, the gene encoding the nucleocapsid protein may be a polynucleotide consisting of a base sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or 100% homology with the base sequence of SEQ ID NO: 17.

[0066] In one specific example, the gene encoding the packaging signal-related protein may be a polynucleotide consisting of a base sequence having 70% or more homology with the base sequence of SEQ ID NO: 19, and specifically, the gene encoding the packaging signal-related protein may be a polynucleotide consisting of a base sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more or 100% homology with the base sequence of SEQ ID NO: 19.

[0067] In one specific example, the respiratory disease-causing virus may be at least one selected from the group consisting of Coronavirus, Human Respiratory Syncytial Virus, Adenovirus, Influenza Virus, Rhinovirus, Enterovirus, Bocavirus, Parainfluenza Virus, Metapneumovirus, Epstein-Barr Virus, Cytomegalovirus, Herpes Simplex Virus, Measles Virus, and Varicella-zoster Virus. Specifically, the virus may be at least one selected from the group consisting of Coronavirus, Human Respiratory Syncytial Virus, Adenovirus, and Influenza Virus, and more specifically, the virus may be Coronavirus.

[0068] The target virus-derived protein or fragment thereof may be a protein or fragment thereof derived from one or more target viruses. Specifically, it may be a protein or fragment thereof derived from one or more respiratory disease-causing viruses. For example, the target protein may be a protein derived from two different viruses (a fusion glycoprotein of RSV and a membrane and envelope protein of SARS-CoV-2), a protein derived from two different virus variants (a spike protein of MERS-CoV and a membrane and envelope protein of SARS-CoV-2; a chimeric spike protein of MERS-CoV and SARS-CoV-2 and a membrane and envelope protein of SARS-CoV-2), etc.

[0069] In one embodiment, a vector in which a sequence encoding the Spike (S), Envelope (E), and Membrane (M) proteins, which are packaging signals and structural proteins of SARS-CoV-2, was inserted instead of a sequence encoding the structural protein of semliki-forest virus (SFV), and a vector in which a sequence encoding the nucleocapsid was inserted were both stably linearized and showed excellent VLP production ability by transfecting cells, thereby confirming the stable VLP production ability of the vector (see Example 1).

[0070] In addition, when a vector having a sequence encoding the Spike (S), Envelope (E), and Membrane (M) proteins, which are the packaging signal and structural proteins of SARS-CoV-2, was transfected into a cell line expressing the nucleocapsid of SARS-CoV-2, it was confirmed that VLPs were stably produced, and thus it was confirmed that a cell line for mass production of the VLPs was also stably produced (see Example 1).

[0071] According to one aspect, the vector, which includes a gene encoding nonstructural proteins of an alphavirus (including NSP1, NSP2, NSP3, NSP4, and RdRp) and a gene encoding a target protein (spike protein, coat protein, membrane protein, and packaging signal-related protein), is manufactured so that the genes can be stably expressed, thereby exhibiting a remarkably excellent VLP production rate. In addition, the vector is conveniently manufactured as a single vector system capable of producing VLPs without a helper vector, and the VLP expressed by the vector can be administered to the mucosa, thereby inducing mucosal immunity and systemic immunity, thereby causing a remarkably excellent immune response, and has the advantage of being excellent in safety because it does not reassemble after a single administration.

[0072]

[0073] Another aspect provides a method for producing cells for producing virus like particles (VLPs), comprising the step of introducing RNA expressed by the vector into the cells.

[0074] The above “vector”, “gene”, “non-structural protein”, “protein”, “respiratory disease causing virus”, “structural protein”, etc. may be within the scope described above.

[0075] The term "RNA" may be used interchangeably with "mRNA" and "saRNA." Specifically, the "RNA" refers to a polymeric material comprising a plurality of ribonucleotide units, specifically, a polymer in which a plurality of ribonucleotide units are linked to each other by phosphodiester bonds of a sugar / phosphate backbone, and the "mRNA" refers to RNA that transfers genetic information to ribosomes in the cytoplasm. For example, it refers to mRNA expressed by the linearized vector. In addition, the "saRNA (self-amplifying RNA)" refers to RNA that includes a sequence encoding an RNA-dependent RNA polymerase so that it can replicate itself within a host cell (Fig. 1). For example, the saRNA refers to mRNA expressed by a linearized vector that includes a sequence encoding an RNA-dependent RNA polymerase of the alpha virus.

[0076] The term "transduction" refers to the transfer of genetic material into cells, and includes transformation, transduction, and transfection. For example, the introduction can be performed by microprojectile bombardment, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, PEG-mediated fusion, microinjection, liposome-mediated methods, etc.

[0077] The term "virus-like particle (VLP)" is generally understood to refer to viral structural proteins that spontaneously self-assemble, mimicking the morphology of actual pathogenic viruses and exhibiting highly similar biochemical properties. Specifically, VLPs are manufactured with a structure similar or identical to that of the original pathogen and can present highly repetitive antigenic epitopes.

[0078] In one specific example, the step of introducing the RNA into the cell may be to simultaneously introduce RNA expressed by a vector comprising a gene encoding a packaging signal-related protein derived from a target virus; the alpha virus-derived polynucleotide; and a gene encoding a second target protein.

[0079] The above "alpha virus-derived polynucleotide" refers to a polynucleotide sequence that is not transcribed into an RNA sequence, or, even if transcribed, is not translated into a protein. For example, it includes a non-transcribed region, an untranslated region, an intergenic region, a regulatory sequence, etc., and specifically, it includes a 3'-UTR sequence, a 5'-UTR sequence, a poly(A) tail sequence, an intron, a centromere, a telomere, an origin of replication (Ori), a promoter, an enhancer, a silencer, etc.

[0080] The above "second target protein" may be a protein derived from a second target virus or a fragment thereof. The second target virus may be identical to or different from the target virus, and the second target virus-derived protein or a fragment thereof may be identical to or different from the target virus-derived protein or a fragment thereof. For example, when the target virus is a coronavirus, the second target virus may be the same coronavirus as the target virus, or may be RSV different from the target virus, and when the target protein is the spike protein of SARS-CoV-2 or a fragment thereof, the second target protein may be the spike protein of SARS-CoV-2 identical to the target protein or a fragment thereof, or may be a fusion glycoprotein of RSV, a spike protein of MERS-CoV, or the like different from the target protein.

[0081] In one specific example, the RNA may include RNA encoding a nonstructural protein of an alphavirus and RNA encoding a protein of interest. Specifically, the RNA may include RNA encoding an RNA-dependent RNA polymerase of an alphavirus and RNA encoding a protein of interest.

[0082] In one specific example, the RNA encoding the target protein is at least one selected from the group consisting of RNA encoding a target virus-derived spike protein or a fragment thereof, RNA encoding a target virus-derived envelope protein or a fragment thereof, RNA encoding a target virus-derived membrane protein or a fragment thereof, and RNA encoding a target virus-derived packaging signal-related protein or a fragment thereof, and the cell may be genetically engineered to express the target virus-derived nucleocapsid protein or a fragment thereof. Specifically, the RNA is at least one selected from the group consisting of RNA encoding a respiratory disease-causing virus-derived spike protein, RNA encoding a respiratory disease-causing virus-derived envelope protein, RNA encoding a respiratory disease-causing virus-derived membrane protein, and RNA encoding a respiratory disease-causing virus-derived packaging signal-related protein, and the cell may be genetically engineered to express the respiratory disease-causing virus-derived nucleocapsid protein. More specifically, the RNA may be an RNA encoding a spike protein derived from a respiratory disease-causing virus, an RNA encoding an envelope protein derived from the respiratory disease-causing virus, an RNA encoding a membrane protein derived from the respiratory disease-causing virus, and an RNA encoding a packaging signal-related protein derived from the respiratory disease-causing virus.

[0083] The term "genetically engineered" refers to the act of introducing one or more genetic modifications into a cell, or a cell produced thereby. For example, the genetically engineered cell can produce or express the nucleocapsid protein of the respiratory disease-causing virus by introducing a vector containing a gene encoding the nucleocapsid protein of the respiratory disease-causing virus.

[0084] According to one aspect of the method, RNA, i.e., saRNA, expressed by a vector including a gene encoding an alphavirus RNA-dependent RNA polymerase (RdRp) and a gene encoding a target protein (spike protein, coat protein, membrane protein, and packaging signal-related protein) is introduced into a cell. The saRNA self-amplifies through RdRp within the cell, expresses the structural proteins of the virus, namely spike, coat, and membrane protein, and is then assembled into a viral particle by the packaging signal to form a VLP containing saRNA therein. Therefore, the VLP containing saRNA produced in the cell does not contain a sequence encoding a viral nucleocapsid within the saRNA, and thus does not cause reinfection after a single infection, so its safety is significantly high.

[0085] In one specific example, the method for producing cells for producing virus-like particles may further include a step of linearizing the vector prior to the step of introducing the RNA into the cell.

[0086] In one specific example, the method for producing cells for producing virus-like particles may further include a step of transcribing the linearized vector into RNA after the linearizing step and before the step of introducing RNA into the cell.

[0087] The above linearization refers to the process of converting the circular or nicked vector into a form with two ends by cleaving it through physical, chemical, or biological methods. Examples include methods of treating with chemicals or restriction enzymes, and linearization through polymerase chain reaction (PCR). In the case of the method for processing the above restriction enzyme, the restriction enzyme may be SpeI, NheI, BamHI, EcoRI, HindIII, kpnI, NotI, PstI, SmaI, XhoI, FokI, Alw26I, BbvI, BsrI, EarI, HphI, MboI, SfaNI, Tth111I, NeaI, NgoMIV, Eco57I, BcgI, BpI, Bsp24I, BaeI, CjeI, EcoPI, StyLTI, Bal31, T4, T7, RecBCD, P25736I, P09030II, P1, DnaseI, Endo R, Cas9, Cpf1, C2c1, C2c2, C2c3, Cas3, Cas3-HD, Cas5, Cas7, Cas8, Cas10, etc., and specifically, the restriction enzyme may be It could be SpeI.

[0088] In one specific example, the cell may be a microorganism, an animal cell, or a plant cell. Specifically, the cell may be a microbial cell such as a yeast, a mold, or a protozoan cell, a plant cell, an insect or amphibian cell, or a mammalian cell such as BHK21, Vero, CHO, HeLa, HEK293, or COS-1.

[0089] In one embodiment, the VLP containing saRNA manufactured through the vector has an atypical structure, and when manufactured using a cell line expressing the nucleocapsid, it was confirmed that a sufficient amount of VLP for vaccination was produced, and when the VLP was inoculated into 2D and 3D cell lines, it was confirmed that the spike protein of SARS-CoV-2 constituting the VLP was expressed within the cells, confirming the infectivity of the VLP. In addition, in the case of the VLP, the shape of the VLP was not confirmed in a microscopic photograph 48 hours after inoculation, confirming that it is a form with excellent safety that does not cause reinfection (see Examples 2 and 3).

[0090] In addition, in another embodiment, when a vector for producing VLPs including saRNA was introduced into cells together with a vector expressing an additional antigen (luciferase), it was confirmed that a VLP capable of delivering not only saRNA but also RNA encoding the additional antigen was packaged together within the produced VLP, and thus, when the additional antigen was delivered together through the VLP, it was confirmed that the expression of the additional antigen could also be promoted without interfering with the expression of saRNA captured within the VLP (see Example 5).

[0091] According to one aspect, cells manufactured according to the method for manufacturing cells for VLP manufacturing can stably mass-produce VLPs, and since the cells themselves express the nucleocapsid of a virus causing a respiratory disease, VLPs containing saRNA produced in the cells do not proliferate further after infecting other cells, thereby manufacturing VLPs with significantly superior safety.

[0092]

[0093] Another aspect provides a cell for producing virus-like particles, manufactured by the method for producing the cell for producing virus-like particles. Specifically, a cell for producing virus-like particles (VLPs) is provided, manufactured by a method for producing a cell for producing virus-like particles, the method comprising a step of introducing RNA expressed by the vector into the cell.

[0094] The above “vector”, “gene”, “non-structural protein”, “protein”, “respiratory disease causing virus”, “structural protein”, “RNA”, “introduction”, etc. may be within the scope described above.

[0095] In one specific example, the RNA encoding the target protein is at least one selected from the group consisting of RNA encoding a target virus-derived spike protein or a fragment thereof, RNA encoding a target virus-derived envelope protein or a fragment thereof, RNA encoding a target virus-derived membrane protein or a fragment thereof, and RNA encoding a target virus-derived packaging signal-related protein or a fragment thereof, and the cell may be genetically engineered to express the target virus-derived nucleocapsid protein or a fragment thereof. Specifically, the RNA is at least one selected from the group consisting of RNA encoding a respiratory disease-causing virus-derived spike protein, RNA encoding a respiratory disease-causing virus-derived envelope protein, RNA encoding a respiratory disease-causing virus-derived membrane protein, and RNA encoding a respiratory disease-causing virus-derived packaging signal-related protein, and the cell may be genetically engineered to express the respiratory disease-causing virus-derived nucleocapsid protein. More specifically, the RNA may be an RNA encoding a spike protein derived from a respiratory disease-causing virus, an RNA encoding an envelope protein derived from the respiratory disease-causing virus, an RNA encoding a membrane protein derived from the respiratory disease-causing virus, and an RNA encoding a packaging signal-related protein derived from the respiratory disease-causing virus.

[0096] In one specific example, the cell may be a microorganism, an animal cell, or a plant cell. Specifically, the cell may be a microbial cell such as a yeast, a mold, or a protozoan cell, a plant cell, an insect or amphibian cell, or a mammalian cell such as BHK21, Vero, CHO, HeLa, HEK293, or COS-1.

[0097] According to one aspect, the cell manufactured according to the method for manufacturing the cell for manufacturing the VLP can stably mass-produce VLP, i.e., efficiently produce an amount of VLP that can be inoculated as a vaccine, and since the cell itself expresses the nucleocapsid of the virus, the saRNA expressed by the vector does not include a sequence encoding the nucleocapsid of the virus, and since the VLP including the saRNA produced in the cell does not further proliferate after infecting another cell, it is possible to manufacture a VLP exhibiting remarkably excellent safety.

[0098]

[0099] Another aspect provides a method for producing virus-like particles, comprising a step of culturing cells for producing the virus-like particles. Specifically, the present invention provides a method for producing virus-like particles, comprising a step of culturing cells for producing virus-like particles (VLPs), the cells being produced by a method for producing cells for producing virus-like particles (VLPs), comprising a step of introducing RNA expressed by the vector into the cells.

[0100] The above “vector”, “gene”, “non-structural protein”, “protein”, “respiratory disease causing virus”, “structural protein”, “RNA”, “introduction”, etc. may be within the scope described above.

[0101] According to one aspect of the manufacturing method, when the cell for manufacturing the virus-like particle is cultured, the RNA expressed by the vector is replicated by RNA-dependent RNA polymerase, and the protein encoded by the RNA is expressed, so that a large amount of VLP is produced in the form of the RNA packaged therein.

[0102] In one specific example, the method for producing the virus-like particle may further include a step of destroying the cell membrane of the cell.

[0103] Specifically, in the method for producing the virus-like particles, the step of destroying the cell membrane includes lysing the cell membrane of the cell to recover the virus-like particles produced within the cell. Specifically, this can be performed using physical, chemical, or biological methods, such as ultrasonic disruption, stirring disruption, homogenizer disruption, repeated freezing and thawing, osmotic shock, use of lytic enzymes, use of organic solvents, use of surfactants, or a combination thereof.

[0104] When the method for producing the above virus-like particles further includes a step of destroying the cell membrane, the method for producing the VLP can produce the VLP at a remarkably high yield.

[0105] In one specific example, the method for producing the virus-like particle may further include a purification step after the step of destroying the cell membrane of the cell.

[0106] Specifically, the term "purification" refers to a method of removing impurities present with the manufactured virus-like particles and increasing their purity so that only virus-like particles remain. For example, the purification may be performed using a method such as filtration, ultrafiltration, centrifugation, chromatography, or a combination thereof.

[0107] Specifically, when the method for producing the virus-like particle further includes the purifying step, the method for producing the VLP can produce the VLP with remarkably high purity.

[0108] Virus-like particles (VLPs) manufactured according to one aspect contain saRNA inside, and since their external shape resembles the structure of a virus that causes a respiratory disease, the VLPs can be administered through the mucosa, which is the infection route of the virus that causes a respiratory disease, and the saRNA inside can self-amplify to produce a sufficient amount of antigen to induce immunity with a single administration. The VLPs induce mucosal immunity and systemic immunity through mucosal administration, resulting in a significantly superior immune response compared to the intramuscular route of administration. In addition, since saRNA does not contain a sequence encoding a nucleocapsid, additional proliferation does not occur, and thus safety is also significantly superior.

[0109] Therefore, the virus-like particles (VLPs) manufactured above can be administered to the mucosa, and after administration, they can induce an immune response (mucosal immunity and systemic immunity) with only a single administration without additional proliferation, thereby producing antibodies within a short period of time. Therefore, when the VLPs are applied to a vaccine, a vaccine with significantly superior immunogenicity and safety compared to an intramuscularly administered vaccine can be manufactured.

[0110]

[0111] Another aspect provides a virus-like particle manufactured by the method for manufacturing the virus-like particle. Specifically, the method for manufacturing the virus-like particle includes a step of culturing a cell for manufacturing the virus-like particle manufactured by a method for manufacturing a cell for manufacturing the virus-like particle, the method including a step of introducing RNA expressed by the vector into the cell, wherein the vector may include a gene encoding a nonstructural protein of an alphavirus and a gene encoding a target protein.

[0112] The above “vector”, “gene”, “non-structural protein”, “protein”, “respiratory disease causing virus”, “structural protein”, “RNA”, “introduction”, “virus-like particle”, etc. may be within the scope described above.

[0113] In one specific example, the target protein may be a target virus-derived protein or a fragment thereof, and specifically may include at least one selected from the group consisting of a target virus-derived spike protein or a fragment thereof, a target virus-derived envelope protein or a fragment thereof, and a target virus-derived membrane protein or a fragment thereof, and more specifically, may be a target virus-derived spike protein or a fragment thereof, a target virus-derived envelope protein or a fragment thereof, and a target virus-derived membrane protein or a fragment thereof, and even more specifically, may be a respiratory disease-causing virus-derived spike protein or a fragment thereof, a respiratory disease-causing virus-derived envelope protein or a fragment thereof, and a respiratory disease-causing virus-derived membrane protein or a fragment thereof.

[0114] The target virus-derived protein or fragment thereof may be a protein or fragment thereof derived from one or more target viruses. Specifically, it may be a protein or fragment thereof derived from one or more respiratory disease-causing viruses. For example, the target protein may be a protein derived from two different viruses (a fusion glycoprotein of RSV and a membrane and envelope protein of SARS-CoV-2), a protein derived from two different virus variants (a spike protein of MERS-CoV and a membrane and envelope protein of SARS-CoV-2; a chimeric spike protein of MERS-CoV and SARS-CoV-2 and a membrane and envelope protein of SARS-CoV-2), etc.

[0115] In one specific example, the RNA may be packaged with the target virus-derived spike protein or a fragment thereof, the target virus-derived envelope protein or a fragment thereof, and the target virus-derived membrane protein or a fragment thereof.

[0116] In one specific example, the RNA may include RNA encoding a nonstructural protein of an alphavirus and RNA encoding a protein of interest. Specifically, the RNA may include RNA encoding an RNA-dependent RNA polymerase of an alphavirus and RNA encoding a protein of interest.

[0117] In one specific example, the RNA encoding the target protein may be at least one selected from the group consisting of RNA encoding a target virus-derived spike protein or a fragment thereof, RNA encoding a target virus-derived envelope protein or a fragment thereof, RNA encoding a target virus-derived membrane protein or a fragment thereof, and RNA encoding a target virus-derived packaging signal-related protein or a fragment thereof. Specifically, the RNA may be at least one selected from the group consisting of RNA encoding a respiratory disease-causing virus-derived spike protein, RNA encoding the respiratory disease-causing virus-derived envelope protein, RNA encoding the respiratory disease-causing virus-derived membrane protein, and RNA encoding the respiratory disease-causing virus-derived packaging signal-related protein. More specifically, the RNA may be RNA encoding a respiratory disease-causing virus-derived spike protein, RNA encoding the respiratory disease-causing virus-derived envelope protein, RNA encoding the respiratory disease-causing virus-derived membrane protein, and RNA encoding the respiratory disease-causing virus-derived packaging signal-related protein.

[0118] In one specific embodiment, the virus-like particle may further comprise RNA expressed by a vector comprising a gene encoding a packaging signal-related protein derived from a target virus; the alpha virus-derived polynucleotide; and a gene encoding a second target protein.

[0119] The above "alpha virus-derived polynucleotide" refers to a polynucleotide sequence that is not transcribed into an RNA sequence, or, even if transcribed, is not translated into a protein. For example, it includes a non-transcribed region, an untranslated region, an intergenic region, a regulatory sequence, etc., and specifically, it includes a 3'-UTR sequence, a 5'-UTR sequence, a poly(A) tail sequence, an intron, a centromere, a telomere, an origin of replication (Ori), a promoter, an enhancer, a silencer, etc.

[0120] The above "second target protein" may be a protein derived from a second target virus or a fragment thereof. The second target virus may be identical to or different from the target virus, and the second target virus-derived protein or a fragment thereof may be identical to or different from the target virus-derived protein or a fragment thereof. For example, when the target virus is a coronavirus, the second target virus may be the same coronavirus as the target virus, or may be RSV different from the target virus, and when the target protein is the spike protein of SARS-CoV-2 or a fragment thereof, the second target protein may be the spike protein of SARS-CoV-2 identical to the target protein or a fragment thereof, or may be a fusion glycoprotein of RSV, a spike protein of MERS-CoV, or the like different from the target protein.

[0121] The virus-like particles may be intended for intraperitoneal, intramucosal, intramuscular, intradermal, subcutaneous, intravenous, intrarectal, intrapulmonary, intrathecal, oral, transdermal or intranasal administration, and specifically may be intended for intramucosal or intranasal administration.

[0122] In one specific example, the virus-like particle can induce a mucosal immune response.

[0123] The term "mucosa" refers to the tissues that line the inner walls of various cavities in the body, comprised of epithelial cells with mucous glands within the skin. Examples of mucosa include the nasal mucosa, nasal mucosa, oral mucosa, ocular mucosa, ear mucosa, genital mucosa, pharyngeal mucosa, tracheal mucosa, bronchial mucosa, lung mucosa, stomach mucosa, intestinal mucosa, and rectal mucosa.

[0124] The term "mucosal immune response" refers to an immune response induced in the mucosa. For example, a mucosal immune response includes antigen-specific immunoglobulin G (IgG) and its subclasses, immunoglobulin A (IgA) and its subclasses, immunoglobulin M (IgM) and its subclasses, and cell-mediated immunity to immunized antigens.

[0125] In one specific example, the virus-like particle can induce at least one selected from the group consisting of a cellular immune response and a humoral immune response at a mucosal surface, and specifically can induce both a cellular immune response and a humoral immune response.

[0126] In one specific example, the virus-like particle can induce at least one selected from the group consisting of a local immune response and a systemic immune response, and specifically can induce both a local immune response and a systemic immune response.

[0127] The virus-like particles according to one aspect are vaccines in a form that can be administered intramucosally or intranasally. They not only induce a mucosal immune response that produces antigen-specific IgA on the mucosal surface, but also simultaneously induce a systemic immune response, resulting in a significantly superior immune response compared to existing intramuscularly administered vaccines, and can induce a rapid immune response. In addition, since vaccination is not performed through a needle, there is no pain or rejection, and it can be administered safely and easily. Specifically, when the virus-like particles are administered intranasally, they can induce a rapid and enhanced immune response against viruses that infect the host through the upper and / or lower respiratory tract mucosal surfaces.

[0128] In one specific example, the mucosal immune response may be one that expresses at least one selected from the group consisting of antigen-specific IgA and IgG in the mucosa, specifically, one that expresses antigen-specific IgA and IgG in the mucosa, and more specifically, one that expresses antigen-specific IgA in the mucosa.

[0129] When the above virus-like particles are administered through the mucosa, which is the primary infection route of the respiratory disease-causing virus, antigen-specific IgA is expressed through the mucosal immune response, and systemic immunity is quickly achieved, resulting in a significantly superior immune response compared to when administered through intramuscular injection, etc., thereby increasing the vaccination efficiency of a vaccine containing the above virus-like particles.

[0130] Virus-like particles manufactured according to this method contain saRNA internally and, because their external shape resembles the structure of a respiratory virus, can infect an individual through the mucous membrane, a route of infection for respiratory viruses. Furthermore, the internal saRNA can self-amplify to produce a sufficient amount of antigen to induce immunity with a single dose. Furthermore, since saRNA does not contain a nucleocapsid, additional proliferation does not occur, resulting in significantly superior safety.

[0131] Therefore, the virus-like particles (VLPs) manufactured above can be administered to the mucosa, and after administration, they can induce an immune response (mucosal immunity and systemic immunity) with only a single administration without additional proliferation, thereby producing antibodies within a short period of time. Therefore, when the VLPs are applied to a vaccine, a vaccine with significantly superior immunogenicity and safety compared to an intramuscularly administered vaccine can be manufactured.

[0132]

[0133] Another aspect provides a vaccine composition for preventing or treating respiratory diseases comprising the virus-like particles.

[0134] The above "vector", "gene", "non-structural protein", "protein", "respiratory disease causing virus", "structural protein", "RNA", "introduction", "virus-like particle", "mucosal", "mucosal immune response", etc. may be within the scope described above.

[0135] The term "vaccine" refers to a biological preparation containing an antigen that induces immunity in a living organism, and refers to an immunogen or antigenic substance that causes immunity in a living organism by administering it to an individual for the purpose of preventing or treating an infection.

[0136] The term "prevention" may mean any act of suppressing or delaying a respiratory disease in an individual by administration of a vaccine composition according to one aspect.

[0137] The term "treatment" may mean any action by which the symptoms of a respiratory disease of an individual are improved or beneficially changed by administration of a vaccine composition according to one aspect.

[0138] The above vaccine composition may be provided comprising one or more immunologically acceptable carriers, excipients or diluents.

[0139] Specifically, the carrier, excipient or diluent may be, for example, glucose, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0140] In one specific example, the vaccine composition may further comprise an adjuvant. The adjuvant may be aluminum hydroxide (Al(OH)3), aluminum phosphate (AlPO4), alum (potassium aluminum sulfate), MF59, virosome, AS04 (a mixture of aluminum hydroxide and monophosphoryl lipid A (MPL)), AS03 (a mixture of DL-α-tocopherol, squalene, and polysorbate 80, an emulsifier), CpG, Flagellin, Poly I:C, AS01, AS02, ISCOMs, or ISCOMMATRIX.

[0141] In one specific example, the immunostimulant may be included in the vaccine composition at 10 to 70 v / v%, specifically 30 to 70 v / v%, more specifically 50 to 70 v / v%. For example, it may be included at 50 to 70 v / v%, 50 to 65 v / v%, 50 to 60 v / v%, 50 to 55 v / v%, 55 to 70 v / v%, 55 to 65 v / v%, 55 to 60 v / v%, 60 to 70 v / v%, 60 to 65 v / v%, or 65 to 70 v / v%.

[0142] The above vaccine composition may be for intraperitoneal, intramucosal, intramuscular, intradermal, subcutaneous, intravenous, intrarectal, intrapulmonary, intrathecal, oral, transdermal or intranasal administration, and specifically may be for intramucosal or intranasal administration.

[0143] The vaccine composition according to one aspect is a vaccine in a form that can be administered intramucosally or intranasally. It not only induces a mucosal immune response that produces antigen-specific IgA on the mucosal surface, but also simultaneously induces a systemic immune response, resulting in a significantly superior immune response compared to existing intramuscularly administered vaccines, and can induce a rapid immune response. In addition, since vaccination is not performed through a needle, there is no pain or rejection, and it can be administered safely and easily. Specifically, when the vaccine composition is administered intranasally, it can induce a rapid and enhanced immune response against viruses that infect the host through the upper and / or lower respiratory tract mucosal surfaces.

[0144] The above vaccine composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, nasal dosage forms such as drips or sprays, and sterile injectable solutions. When formulating, diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants can be used.

[0145] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations can be prepared by mixing the lecithin-like emulsifier with at least one excipient, such as starch, calcium carbonate, glucose, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration can include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous preparations, suspensions, emulsions, and lyophilized preparations. Non-aqueous preparations and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suitable penetrants for intranasal administration are generally known to those skilled in the art, and suitable formulations may be formulated to be sterile, isotonic, and buffered to ensure stability and compliance. Intranasal preparations are also formulated to stimulate mucus secretion in various ways to maintain normal ciliary function, maintain a pH of 5.5 to 6.5, and may include antimicrobial preservatives and suitable stabilizers.

[0146] In one specific example, the respiratory disease may be one or more viral infections selected from the group consisting of coronavirus infection, human respiratory syncytial virus infection, adenovirus infection, influenza virus infection, rhinovirus infection, enterovirus infection, bocavirus infection, parainfluenza virus infection, metapneumovirus infection, Epstein-Barr virus infection, cytomegalovirus infection, herpes simplex virus infection, measles virus infection, and varicella-zoster virus infection.

[0147] The above vaccine composition is administered in an immunologically effective amount. The term "immunologically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration and the excretion rate, the duration of treatment, concomitant drugs, and other factors well known in the medical field. The administration may be administered once a day or in several divided doses. For example, the vaccine composition may be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more, and the administration may be administered at intervals of about 1 to 16 weeks, about 1 to 12 weeks, about 1 to 8 weeks, or about 1 to 4 weeks. If the above vaccine composition causes a second or more infection by the virus it targets, it may be re-administered regularly.

[0148] The term "administration" above refers to introducing a given substance into an individual through an appropriate method, and "individual" refers to any living organism, including rats, mice, pigs, horses, cows, and livestock, including humans, that may have a respiratory disease. A specific example may be a mammal, including humans.

[0149] A single dose of the above vaccine composition may be 0.1 μL to 0.6 mL, specifically 10 μL to 0.6 mL, more specifically 30 μL to 0.6 mL.

[0150] The vaccine composition comprises 5 ㎍ to 5000 ㎍, 5 ㎍ to 2500 ㎍, 5 ㎍ to 1000 ㎍, 5 ㎍ to 500 ㎍, 5 ㎍ to 250 ㎍, 5 ㎍ to 100 ㎍, 5 ㎍ to 50 ㎍, 50 ㎍ to 5000 ㎍, 50 ㎍ to 2500 ㎍, 50 ㎍ to 1000 ㎍, 50 ㎍ to 500 ㎍, 50 ㎍ to 250 ㎍, 50 ㎍ to 100 ㎍, 100 ㎍ to 5000 ㎍, 100 ㎍ to 2500 ㎍, 100 ㎍ to 1000 ㎍, 100 ㎍ to 500 ㎍, 100 ㎍ to 250 ㎍, 250 ㎍ to 5000 ㎍, 250 ㎍ to 2500 ㎍, 250 ㎍ to 1000 ㎍, 250 ㎍ to 500 ㎍, 500 ㎍ to 5000 ㎍, 500 ㎍ to 2500 ㎍, 500 ㎍ to 1000 ㎍, 1000 ㎍ to 5000 ㎍, 1000 ㎍ to 2500 ㎍ or 2500 ㎍ to 5000 ㎍.

[0151] The above vaccine composition may be administered as a booster vaccine. "Booster" refers to a second antigenic stimulus administered after the first antigenic stimulus during vaccination with a vaccine, and when antibodies are produced, the second antigenic stimulus achieves a faster and higher antibody production rate than the response that occurs after the first antigenic stimulus. The "booster vaccine" is administered after the first or second dose of the above vaccine composition, and may correspond to, for example, the third dose. It broadly refers to a dose that can exhibit an immune-enhancing effect after the first dose.

[0152] In one specific example, the respiratory disease may be at least one selected from the group consisting of respiratory inflammatory lung disease, chronic obstructive pulmonary disease (COPD), sinusitis, allergic rhinitis, lower respiratory tract infection, tracheitis, acute bronchitis, chronic bronchitis, emphysema, pneumonia, asthma, bronchiectasis, emphysema, pulmonary tuberculosis, acute respiratory distress syndrome, diffuse interstitial lung disease, cystic fibrosis, otitis media, bronchiolitis, and pulmonary fibrosis.

[0153] In one specific example, the vaccine composition can induce a mucosal immune response.

[0154] In one specific example, the vaccine composition can induce at least one selected from the group consisting of a cellular immune response and a humoral immune response at a mucosal surface, and specifically can induce both a cellular immune response and a humoral immune response.

[0155] In one specific example, the vaccine composition can induce at least one selected from the group consisting of a local immune response and a systemic immune response, and specifically can induce both a local immune response and a systemic immune response.

[0156] In one specific example, the mucosal immune response may be one that expresses at least one selected from the group consisting of antigen-specific IgA and IgG in the mucosa, specifically, one that expresses antigen-specific IgA and IgG in the mucosa, and more specifically, one that expresses antigen-specific IgA in the mucosa.

[0157] When the above vaccine composition is administered through the mucosa, which is the primary infection route of the respiratory disease-causing virus, antigen-specific IgA is expressed through the mucosal immune response, and systemic immunity is quickly achieved, resulting in a significantly superior immune response compared to when the vaccine is administered through intramuscular injection, etc., thereby increasing the vaccination efficiency of the vaccine.

[0158] In one example, when VLPs containing saRNA were intranasally administered to mice, the titers of IgG and IgA, which are mucosal immune antibodies, were significantly higher, and the titers of neutralizing antibodies, secretion of INF-γ, expression of INF-α and INF-β genes, and the number of T cells were also significantly better than those of the control group, intramuscular administration group, and LNP nasal administration group.

[0159] In addition, when the VLP containing the saRNA was intranasally administered to mice when a virus reinfection occurred after vaccination, the body weight, survival rate, virus titer in the lungs and mucosa, and lung function of the mice were restored to normal ranges, which was significantly superior to the intramuscular administration group and the LNP nasal administration group, confirming that the nasal administration of the VLP exhibited excellent immunogenicity and protective ability against reinfection (see Example 4).

[0160] In one embodiment, in the case of the above vector system, it was confirmed that even when a sequence portion encoding a structural protein of SARS-CoV-2 was inserted into another foreign gene, it was possible to stably produce it, and that a vector expressing an additional antigen could be simultaneously transfected into cells together with the vector, thereby producing a VLP containing an additional antigen RNA together with saRNA. Chimeric VLPs in which the receptor binding site of the VLP was changed were also stably produced, and it was confirmed that the chimeric VLPs also exhibited remarkably excellent immunogenicity and protective ability, confirming that the vector system is useful as a vaccine template for various viruses (see Example 5).

[0161] According to one aspect, the vaccine composition can be administered through the mucosa, the primary route of infection for the virus. Since the saRNA contained within the VLPs in the vaccine composition self-amplifies, a sufficient amount of antibodies can be generated with just a single administration. Furthermore, the VLPs do not undergo additional proliferation, ensuring excellent safety. Furthermore, since they induce mucosal immunity, mucosal-specific antibodies are produced, leading to a significantly superior immune response compared to intramuscular injection.

[0162]

[0163] Another aspect provides a method for preventing or treating a respiratory disease, comprising administering to a subject in need thereof a vaccine composition comprising the virus-like particles.

[0164] The above “vector”, “non-structural protein”, “respiratory disease causing virus”, “structural protein”, “RNA”, “introduction”, “virus-like particle”, “administration”, etc. may be within the scope described above.

[0165] In one specific example, the respiratory disease may be one or more viral infections selected from the group consisting of coronavirus, human respiratory syncytial virus, adenovirus, influenza virus, rhinovirus, enterovirus, bocavirus, parainfluenza virus, metapneumovirus, Epstein-Barr virus, cytomegalovirus, herpes simplex virus, measles virus, and varicella-zoster virus.

[0166] In the above administration step, the vaccine composition may be administered at a time in an amount of 0.1 μL to 0.6 mL, specifically 10 μL to 0.6 mL, and more specifically 30 μL to 0.6 mL.

[0167] The vaccine composition comprises 5 ㎍ to 5000 ㎍, 5 ㎍ to 2500 ㎍, 5 ㎍ to 1000 ㎍, 5 ㎍ to 500 ㎍, 5 ㎍ to 250 ㎍, 5 ㎍ to 100 ㎍, 5 ㎍ to 50 ㎍, 50 ㎍ to 5000 ㎍, 50 ㎍ to 2500 ㎍, 50 ㎍ to 1000 ㎍, 50 ㎍ to 500 ㎍, 50 ㎍ to 250 ㎍, 50 ㎍ to 100 ㎍, 100 ㎍ to 5000 ㎍, 100 ㎍ to 2500 ㎍, 100 ㎍ to 1000 ㎍, 100 ㎍ to 500 ㎍, 100 ㎍ to 250 ㎍, 250 ㎍ to 5000 ㎍, 250 ㎍ to 2500 ㎍, 250 ㎍ to 1000 ㎍, 250 ㎍ to 500 ㎍, 500 ㎍ to 5000 ㎍, 500 ㎍ to 2500 ㎍, 500 ㎍ to 1000 ㎍, 1000 ㎍ to 5000 ㎍, 1000 ㎍ to 2500 ㎍ or 2500 ㎍ to 5000 ㎍.

[0168] The vaccine composition used in the method for preventing or treating respiratory diseases according to one aspect can be administered through the mucosa, which is the primary infection route of the virus, and since the saRNA within the VLP contained in the vaccine composition self-amplifies, a sufficient amount of antibodies can be generated with just a single administration. In addition, the VLP does not undergo additional proliferation, so it is excellent in safety, and since it induces mucosal immunity, mucosal-specific antibodies are produced, inducing a significantly superior immune response compared to intramuscular injection.

[0169]

[0170] Another aspect provides the use of the virus-like particles for the manufacture of a medicament for preventing or treating the above respiratory disease.

[0171] The above “vector”, “non-structural protein”, “respiratory disease causing virus”, “structural protein”, “RNA”, “introduction”, “virus-like particle”, etc. may be within the scope described above.

[0172] In one specific example, the respiratory disease may be one or more viral infections selected from the group consisting of coronavirus, human respiratory syncytial virus, adenovirus, influenza virus, rhinovirus, enterovirus, bocavirus, parainfluenza virus, metapneumovirus, Epstein-Barr virus, cytomegalovirus, herpes simplex virus, measles virus, and varicella-zoster virus.

[0173] A single dose of the above respiratory disease prevention or treatment agent may be administered in an amount of 0.1 μL to 0.6 mL, specifically 10 μL to 0.6 mL, and more specifically 30 μL to 0.6 mL.

[0174] The virus-like particles in a single dose of the above respiratory disease prevention or treatment drug are 5 ㎍ to 5000 ㎍, 5 ㎍ to 2500 ㎍, 5 ㎍ to 1000 ㎍, 5 ㎍ to 500 ㎍, 5 ㎍ to 250 ㎍, 5 ㎍ to 100 ㎍, 5 ㎍ to 50 ㎍, 50 ㎍ to 5000 ㎍, 50 ㎍ to 2500 ㎍, 50 ㎍ to 1000 ㎍, 50 ㎍ to 500 ㎍, 50 ㎍ to 250 ㎍, 50 ㎍ to 100 ㎍, 100 ㎍ to 5000 ㎍, 100 ㎍ to 2500 ㎍, 100 ㎍ to 1000 ㎍, 100 ㎍ to 500 ㎍, 100 ㎍ to 250 ㎍, 250 ㎍ to 5000 ㎍, 250 ㎍ to 2500 ㎍, 250 ㎍ to 1000 ㎍, 250 ㎍ to 500 ㎍, 500 ㎍ to 5000 ㎍, 500 ㎍ to 2500 ㎍, 500 ㎍ to 1000 ㎍, 1000 ㎍ to 5000 ㎍, 1000 ㎍ to 2500 ㎍ or 2500 ㎍ to 5000 ㎍.

[0175] The above virus-like particles for the manufacture of a drug for the prevention or treatment of respiratory diseases according to the aspect can be administered through the mucosa, which is the primary infection route of the virus, and since the saRNA within the VLP self-amplifies, a sufficient amount of antibodies can be produced with just a single administration. In addition, the VLP does not undergo additional proliferation, so it is excellent in safety, and since it induces mucosal immunity, mucosal-specific antibodies are produced, inducing a significantly superior immune response compared to when the VLP is injected intramuscularly.

[0176]

[0177] The present invention relates to a vector for producing a virus-like particle (VLP), a VLP produced by the vector, and a vaccine composition comprising the same. The vector can easily and conveniently produce a VLP containing saRNA without a helper vector using a single vector system, and the VLP produced by the vector and the vaccine composition comprising the same can be administered through the mucosa, which is the primary infection route of the virus, and since the saRNA in the VLP self-amplifies, a sufficient amount of antibodies can be produced with just a single administration. In addition, the VLP does not undergo additional proliferation and thus has excellent safety, and as a form that can be administered to the mucosa, it can induce mucosal immunity and exhibit a significantly superior effect in preventing or treating respiratory diseases compared to therapeutic agents administered intramuscularly.

[0178]

[0179] Figure 1 is a diagram showing expression when existing mRNA and self-amplifying RNA are inoculated into the body.

[0180] Figure 2 is a diagram showing the structure of a vector for manufacturing VLP containing saRNA, RNA expressed by the vector, and the manufactured VLP.

[0181] Figure 3 is a diagram showing the composition of a vector for manufacturing VLPs containing saRNA.

[0182] Figure 4 is a diagram showing the results of a Western blot of RNAs transcribed by each vector into which each gene encoding the spike, envelope, membrane, and nucleocapsid of SARS-CoV-2 was inserted, and a Western blot of RNAs expressed by a vector into which the genes encoding the spike, envelope, and membrane of SARS-CoV-2 were inserted and a vector into which the genes encoding the nucleocapsid were inserted.

[0183] Figure 5 is a diagram showing the results of confirming the produced protein through an immunofluorescence assay after transfecting cells with RNA produced by linearizing a vector.

[0184] Figure 6 is a diagram showing the results of Western blotting performed on the lysate and supernatant of transfected cells to confirm the production of VLPs.

[0185] Figure 7 is a diagram confirming whether VLPs were produced in the HEK 293-N cell line.

[0186] Figure 8 is a diagram showing the results of a Western blot of RNA transcribed from a vector into which genes encoding the spike, envelope, and membrane of SARS-CoV-2 are inserted.

[0187] Figure 9 is a diagram showing the results of immunofluorescence assay for the protein produced after transfecting saRNA into HEK 293-N cell lines.

[0188] Figure 10 is a diagram showing the results of Western blotting performed on the lysate and supernatant of transfected cells to confirm the production of VLPs.

[0189] Figure 11 is a micrograph of a VLP mass production cell line expressing nucleocapsid.

[0190] Figure 12 is a TEM photograph showing the structure of VLP.

[0191] Figure 13 is a diagram showing the particle size distribution of VLPs.

[0192] Figure 14 is a diagram confirming VLP infectivity in BHK-21, HACE2-293T, and A549-HACE2-TMPRSS2 cell lines.

[0193] Figure 15 is a diagram showing the VLP infectivity in HACE2-293T and A549-HACE2-TMPRSS2 cell lines confirmed through microscopic photographs.

[0194] Figure 16 is a diagram confirming the expression of the spike protein of SARS-CoV-2 in human bronchial epithelial cells through an immunofluorescence assay.

[0195] Figure 17 is a diagram confirming that the spike gene of SARS-CoV-2 was expressed in human bronchial epithelial cells through ISH.

[0196] Figure 18 is a diagram showing the results of Western blot performed on lysates of cells infected with VLP.

[0197] Figure 19 shows TEM images of cells 30 minutes and 48 hours after VLP infection of VERO E6 cells.

[0198] Figure 20 is a diagram confirming the expression of the spike gene of SARS-CoV-2 in the lungs of mice after intranasal inoculation with VLP.

[0199] Figure 21 is a diagram confirming the expression of membrane proteins of SARS-CoV-2 in the lungs of mice after nasal inoculation with VLP.

[0200] Figure 22 is a schematic diagram illustrating a method for administering a VLP vaccine to BALB / c mice.

[0201] Figure 23 is a diagram showing the α-spike RBD IgG and α-spike trimer IgG titers in mice after VLP vaccination.

[0202] Figure 24 is a diagram showing the α-spike RBD IgA and α-spike trimer IgA titers in mice after VLP vaccination.

[0203] Figure 25 is a diagram showing the neutralizing antibody titer in mice after VLP vaccination.

[0204] Figure 26 is a diagram showing the amount of INF-γ secreted in mice after VLP vaccination.

[0205] Figure 27 is a diagram showing the expression levels of INF-α and INF-β genes in mice after VLP vaccination.

[0206] Figure 28 is a diagram showing the number of T cells in mice after VLP vaccination.

[0207] Figure 29 is a diagram showing the change in body weight of mice after VLP vaccination and reinfection with the virus.

[0208] Figure 30 is a diagram showing the survival rate of mice after being reinfected with a virus following VLP vaccination.

[0209] Figure 31 is a diagram showing changes in the titer of the virus present in the lungs of mice after VLP vaccination and reinfection with the virus.

[0210] Figure 32 is a diagram showing changes in lung function in mice after VLP vaccination and reinfection with the virus.

[0211] Figure 33 is a schematic diagram illustrating a method for administering a VLP vaccine to hACE2-Tg mice.

[0212] Figure 34 is a diagram showing the α-spike RBD IgG and α-spike trimer IgG titers in mice after VLP vaccination.

[0213] Figure 35 is a diagram showing the α-spike RBD IgA and α-spike trimer IgA titers in mice after VLP vaccination.

[0214] Figure 36 is a diagram showing the neutralizing antibody titer in mice after VLP vaccination.

[0215] Figure 37 is a diagram showing the amount of INF-γ secreted in mice after VLP vaccination.

[0216] Figure 38 is a diagram showing the expression levels of INF-α and INF-β genes in mice after VLP vaccination.

[0217] Figure 39 is a diagram showing changes in body weight in mice after VLP vaccination.

[0218] Figure 40 is a diagram showing the change in body weight of mice after VLP vaccination and reinfection with the virus.

[0219] Figure 41 is a diagram showing the survival rate of mice after reinfection with the virus following VLP vaccination.

[0220] Figure 42 is a diagram showing the results of measuring the virus present in the lungs and nasal cavity of mice after reinfection with the virus following VLP vaccination using qPCR.

[0221] Figure 43 is a diagram showing changes in lung function in mice after VLP vaccination and reinfection with the virus.

[0222] Figure 44 is a diagram confirming the expression of a foreign gene protein in a cell of a vector into which the foreign gene LacZ has been inserted.

[0223] Figure 45 is a schematic diagram showing the composition of each vector and the structure of the manufactured VLP when a vector expressing an additional antigen (luciferase) is introduced into cells together with a vector system for VLP production.

[0224] Figure 46 is a diagram showing the expression levels of NSP1, spike, and luciferase genes when a vector expressing an additional antigen (luciferase) was introduced into cells together with a vector system for VLP production.

[0225] Figure 47 is a photograph confirming the expression of additional antigens in mice infected with SARS2 JN.1 virus and uninfected mice when a vector expressing an additional antigen (luciferase) was introduced into cells together with a vector system for VLP production.

[0226] Figure 48 is a graph confirming the expression of additional antigens in mice infected with SARS2 JN.1 virus and uninfected mice when a vector expressing an additional antigen (luciferase) was introduced into cells together with a vector system for VLP production.

[0227] Figure 49 is a diagram showing a VLP generated by changing the RBD of the spike protein of SARS-CoV-2 Omicron BA.1 to the RBD of SARS-CoV-2 Omicron XBB.

[0228] Figure 50 is a diagram showing a VLP generated by changing the RBD of the spike protein of SARS-CoV-2 Omicron BA.1 to the RBD of MERS-CoV.

[0229] Figure 51 is a diagram confirming the production of MERS-CoV chimeric VLPs in a cell line for mass production.

[0230] Figure 52 is a diagram confirming the amount of genes expressing the envelope and membrane proteins of MERS-CoV chimeric VLP expressed in a cell line for mass production of VLP.

[0231] Figure 53 is a schematic diagram illustrating a method for administering the Omicron XBB.1 chimeric VLP vaccine to BALB / c mice.

[0232] Figure 54 is a diagram showing the neutralizing antibody titer in mice after vaccination with the Omicron XBB.1 chimeric VLP vaccine.

[0233] Figure 55 is a diagram showing the expression levels of INF-α and INF-β genes in mice after vaccination with the Omicron XBB.1 chimeric VLP vaccine.

[0234] Figure 56 is a diagram showing changes in the titer of the virus present in the lungs of mice after reinfection with the virus following vaccination with the Omicron XBB.1 chimeric VLP vaccine.

[0235] Figure 57 is a schematic diagram illustrating the experimental method for administering the Omicron XBB.1 chimeric VLP vaccine to hACE2-Tg mice.

[0236] Figure 58 is a diagram showing the neutralizing antibody titer in mice after vaccination with the Omicron XBB.1 chimeric VLP vaccine.

[0237] Figure 59 is a diagram showing the expression levels of INF-α and INF-β genes in mice after vaccination with the Omicron XBB.1 chimeric VLP vaccine.

[0238] Figure 60 is a diagram showing the change in body weight of mice after vaccination with the Omicron XBB.1 chimeric VLP vaccine and then reinfection with the virus.

[0239] Figure 61 is a diagram showing the change in survival rate of mice after reinfection with the virus following vaccination with the Omicron XBB.1 chimeric VLP vaccine.

[0240] Figure 62 is a diagram showing changes in the titer of the virus present in the lungs of mice after reinfection with the virus following vaccination with the Omicron XBB.1 chimeric VLP vaccine.

[0241] Figure 63 is a diagram confirming the RBD protein of MERS-CoV expressed when MERS-CoV chimeric VLP was inoculated into VERO E6 cell line.

[0242] Figure 64 is a schematic diagram illustrating a method for administering a MERS-CoV chimeric VLP vaccine to hDPP4 mice.

[0243] Figure 65 is a diagram showing the IgG titer in mice after vaccination with the MERS-CoV chimeric VLP vaccine.

[0244] Figure 66 is a diagram showing neutralizing antibody titers in mice after vaccination with the MERS-CoV chimeric VLP vaccine.

[0245] Figure 67 is a diagram showing the change in body weight of mice after reinfection with the virus following vaccination with the MERS-CoV chimeric VLP vaccine.

[0246] Figure 68 is a diagram showing the survival rate of mice after reinfection with the virus following vaccination with the MERS-CoV chimeric VLP vaccine.

[0247] Figure 69 is a diagram showing changes in the titer of the virus present in the lungs of mice after reinfection with the virus following vaccination with the MERS-CoV chimeric VLP vaccine.

[0248]

[0249] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0250]

[0251] Reference example

[0252] <Reference Example 1> SARS-CoV-2 VLP infection

[0253] VERO E6, HEK-293T, A549-HACE2-TMPRSS2, and BHK-21 cells were infected with 20-fold diluted VLPs (SARS-CoV-2 or MERS), and after 1 h, the medium was replaced with DMEM containing 2% FBS and maintained for 48 h. At 48 h post-infection (hpi), cells were washed three times with warm PBS, and viral and host RNAs were extracted from cell lysates and supernatants using a Maxwell RSC Viral Total Nucleic Acid Purification Kit (Promega, WI).

[0254]

[0255] <Reference Example 2> Quantitative RT-PCR

[0256] RNA yield was assessed by RT-qPCR analysis. Specifically, cDNA was amplified in the presence of primers specifically targeting internal viral sequences common to all viral transcripts (N amplicon) or subgenomic N mRNA in a reaction mixture containing the 1× One Step TB Green PrimeScript RT-PCR kit (Takara) on a StepOnePlus Real-Time PCR system (QuantStudio 5; Thermo Fisher Scientific). The reaction was performed at 42°C for 5 min, 95°C for 10 s, and then 40 cycles of 95°C for 5 s and 60°C for 30 s.

[0257]

[0258] <Reference Example 3> ELISA and neutralizing antibody test

[0259] 96-well NUNC-MaxiSorp plates were coated overnight at 4°C with SARS-CoV-2 Omicron BA.1 RBD (Sinobiological, #40592-V08H121) or full-length spike trimer (Sinobiological, #40589-V08H26), diluted to 1 μg / mL in phosphate-buffered saline (PBS), pH 7.4. Plates were blocked for 1 h at 37°C with blocking buffer (PBS containing 5% BSA and 0.05% tween-20). Samples were serially diluted starting at a 1:10 dilution with reagent diluent (PBS containing 1% BSA and 0.05% tween-20). After incubation at 37°C for 2 h, the plates were washed four times with washing buffer (PBS containing 0.05% Tween-20), and then 1:20,000 diluted goat anti-mouse IgG-HRP (abcam, #ab97023) was added and incubated at 37°C for 1 h.

[0260] Serum neutralization test was performed with Vero E6 cells dissolved in DMEM (supplemented with 10% FBS, 1% L-glutamine, 100 U / ml penicillin-streptomycin) seeded at 3 × 10 per well in 96-well plates. 4 The cells were seeded at a density of 100 µg / well. After 24 hours, the serum was inactivated by incubation at 56°C for 30 minutes, then diluted 1:10 in DMEM (without FBS, 1% L-glutamine, and 100 U / ml penicillin-streptomycin), and serially diluted 1:2 in 96-well plates. Then, 100 TCID per well 50 An equal volume of SARS-CoV-2 consisting of was added to the diluted serum. The serum-virus mixture was incubated at 37°C for 1 hour and then added to Vero E6 cells. The Vero E6 culture medium was then replaced with 150 μL of low-serum DMEM (containing 2% FBS, 1% L-glutamine, and 100 U / ml penicillin-streptomycin) and incubated at 37°C for 72 hours to observe cytopathic effects.

[0261]

[0262] <Reference Example 4> Animal testing

[0263] Animal experiments were performed with the approval of the Animal Experiment Ethics Committee of the Institute for Basic Science (IBS-2023-039). To investigate the efficacy of SARS-CoV-2 VLPs (XBB.1 chimeric VLPs or BA.1 VLPs) as a vaccine, 8- to 10-week-old female BALB / c or hACE2-Tg mice were randomly divided into groups of 13 each, and to investigate the efficacy of MERS VLPs as a vaccine, 8- to 10-week-old female Hddp4 mice were randomly divided into groups of 10 each. After anesthetizing the mice, they were intranasally inoculated with 30 μL of VLPs or PBS.

[0264] For SARS-CoV-2 VLP, 1 LD was administered to mice 4 weeks after vaccination. 50Wuhan MA or XBB.1 was intranasally inoculated with 30 μL, and body weight and survival rate were observed for approximately 14 days. Two mice per group were euthanized at 3, 5, and 7 dpi (days post infection), and nasal conchae and lung tissues were obtained for histopathological examination and qRT-PCR.

[0265] For MERS VLP, boosting MERS VLP was administered 5 weeks after vaccination, and 2 weeks after boosting vaccination, 10 LD was administered to mice. 50 MERS-CoV was intranasally inoculated with 30 μL, and body weight and survival rate were observed for approximately 14 days. Two mice per group were euthanized at 3, 5, and 7 dpi, and nasal conchae and lung tissues were obtained for histopathological examination and qRT-PCR.

[0266]

[0267] <Reference Example 5> Lung histopathology

[0268] Immunohistochemistry (IHC) was performed using a Bond RXm (Leica) automated platform. Specifically, mouse lung slides were dewaxed, rehydrated, and pretreated with epitope retrieval ER2 high-pH buffer (Leica). Endogenous peroxidase was inactivated with H2O2, and nonspecific signals were blocked with mouse serum (1 / 500 dilution). Rabbit anti-SARS-CoV-2 nucleocapsid (Abcam, #ab271180) primary antibody was used at a concentration of 1:200, followed by peroxidase-conjugated anti-rabbit IgG (Jackson ImmunoResearch, #111-035-003) at a dilution of 1:1000.

[0269] Immunoreactivity was visualized using diaminobenzidine chromogen reaction. Slides were counterstained with hematoxylin, dehydrated serially with ethanol, cleared with xylene, and permanently mounted. Negative controls were prepared by replacing the primary antibody with BOND primary antibody diluent (Leica, #AR9352). Photomicrographs at 40x magnification were taken using an Axio Scan Z1.

[0270] In situ hybridization (ISH) was performed using the RNAscope 2.5 HD Detection Reagent-RED Kit (ACDBio, MN) according to the manufacturer's instructions. Specifically, lung slides were deparaffinized, treated with H2O2, and pretreated with antigen retrieval solution. The nCoV2019-S (ACDBio, #848561) probe was hybridized for 2 hours. The signal was amplified with Amp 1-6 solution and detected using RED solution. The slides were counterstained with hematoxylin, mounted, and images were captured at 40x magnification using an Axio Scan Z1.

[0271]

[0272] Manufacturing example

[0273] <Manufacturing Example 1> Manufacturing of vectors

[0274] The pSP6-26s plasmid was constructed by deleting the nonstructural and structural proteins of Semliki-forest virus (SFV) from the pSFV3 (Addgene, #92072) backbone. Sequences encoding the structural proteins and the packaging signal of SARS-CoV-2 omicron BA.1 were synthesized (Bionics, Korea) and subcloned into the pSP6-26s and pcDNA3.1(+) (Genscript, NJ) plasmids. The replicon plasmid pSFV3-SEM-PS9 was constructed by subcloning the structural proteins (spike protein, coat protein, and membrane protein) and the packaging signal sequences of SARS-CoV-2 omicron BA.1 into the pSFV3 backbone (including the nonstructural proteins NSP1 to NSP4 of Semliki-forest virus).

[0275] For the vector producing chimeric VLP, only the RBD (Receptor binding domain) portion of the spike protein of SARS-CoV-2 Omicron BA.1 in the above pSFV3-SEM-PS9 was changed to the RBD of SARS-CoV-2 Omicron XBB.1 or MERS-CoV, respectively.

[0276] For the packaging signal sequence of the above SARS-CoV-2, the sequence of SEQ ID NO: 20 or a gene sequence encoding a portion of NSP11 to NSP12 of the SARS-CoV-2 BA.1 strain was used. The inserted sequences are as shown in Table 1 below, and the structure of the vector is as shown in Fig. 3.

[0277] Sequence numberSequence nameSequence number 1Semrimki Forest Virus (SFV) NSP1(DNA)Sequence number 2SFV NSP1(RNA)Sequence number 3SFV NSP2(DNA)Sequence number 4SFV NSP2(RNA)Sequence number 5SFV NSP3(DNA)Sequence number 6SFV NSP3(RNA)Sequence number 7SFV NSP4(DNA)Sequence number 8SFV NSP4(RNA)Sequence number 9SFV RdRp(DNA)Sequence number 10SFV RdRp(RNA)Sequence number 11SARS-CoV-2(BA.1) Spike gene(DNA)Sequence number 12SARS-CoV-2(BA.1) Spike gene(RNA)Sequence number 13SARS-CoV-2(BA.1) Envelope gene(DNA)Sequence number 14SARS-CoV-2(BA.1) Envelope gene(RNA)Sequence number 15SARS-CoV-2(BA.1) membrane gene (DNA) SEQ ID NO: 16SARS-CoV-2(BA.1) membrane gene (RNA) SEQ ID NO: 17SARS-CoV-2(BA.1) nucleocapsid gene (DNA) SEQ ID NO: 18SARS-CoV-2(BA.1) nucleocapsid gene (RNA) SEQ ID NO: 19SARS-CoV-2(BA.1) packaging signal 1 (DNA) SEQ ID NO: 20SARS-CoV-2(BA.1) packaging signal 1 (RNA) SEQ ID NO: 21saRNA expressed by pSFV3-SEM-PS9 SEQ ID NO: 22pSFV3-SEM-PS9 SEQ ID NO: 23SARS-CoV-2(XBB.1 RBD) chimeric spike gene (DNA) SEQ ID NO: 24SARS-CoV-2(XBB.1 RBD) chimeric spike Gene (RNA) Sequence number 25 MERS-CoV (MERS RBD) chimeric spike gene (DNA) Sequence number 26 MERS-CoV (MERS RBD) chimeric spike gene (RNA)

[0278]

[0279] <Manufacturing Example 2> Cell culture for VLP production

[0280] HEK 293 cells (Homo sapienskidney epithelial, ATCC: CRL-1573) were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific) and the antibiotics penicillin (100 U / mL) and streptomycin (100 μg / mL). Cells were maintained at 37°C in a 5% CO2 atmosphere.

[0281] HEK 293 (HEK 293-N) cells expressing nucleocapsid (N) were obtained by transfection with linearized pcDNA3.1(+)-N plasmid using Lipofectamine 3000 (Thermo Fisher Scientific). After 2 days, cells were cloned with 1 mg / ml of G418 (InvivoGen), and nucleocapsid expression was confirmed by immunofluorescence analysis.

[0282]

[0283] <Manufacturing Example 3> Manufacturing of SARS-CoV-2 VLPs containing saRNA

[0284] RNA transcripts were obtained through in vitro transcription using the MEGAscript SP6 kit (Invitrogen). Then, HEK 293-N cells (8 × 10 6 400 μg of replicon RNA was added to a 4 mm cuvette containing a solution containing cells. A single electric pulse was applied using a GenePulser X (Bio-Rad) set to 260 V and 550 μF. After transferring the cells to a 100 mm culture dish, VLPs were prepared. 48 hours after transfection, the culture medium containing VLPs was obtained and centrifuged at 3,000 rpm for 10 min. The supernatant was filtered through a 0.45 μm syringe filter.

[0285] To concentrate and purify SARS-CoV-2 VLPs, the filtrate was loaded onto a 20% sucrose cushion and ultracentrifuged at 30,000 rpm for 4 h at 4 °C using an SW32Ti rotor (Beckman Coulter). The resulting pellet was then suspended in a PBS solution, and the remaining sucrose and proteins were removed by centrifugation at 4,000 rpm for 1 h at 4 °C using an Amicon Ultra-2mL-30K (Merck). The purified VLPs were finally suspended in PBS and stored at -80 °C until use.

[0286]

[0287] Example

[0288] <Example 1> Confirmation of production of VLP containing saRNA

[0289] When a vector for producing VLPs into which the structural protein and packaging sequences of SARS-CoV-2 are inserted is infected into cells, to determine whether the vector can stably produce VLPs, i.e., whether saRNA (self-amplifying RNA) is transcribed and VLPs are produced according to the sequence inserted into the vector. In order to confirm this, according to Manufacturing Examples 1 to 3, a replicon plasmid vector (pSFV3-SEM-PS9, Fig. 3) was transcribed in vitro (IVT) to produce saRNA, which was then inoculated into a cell line to produce VLPs (Fig. 2), and the produced VLPs were confirmed through Western blot and immunofluorescence assay.

[0290]

[0291] (1) Confirmation of the stability of the vector system in the BHK-21 cell line

[0292] As shown in Fig. 4, instead of the sequence encoding the structural proteins of Semliki-forest virus (SFV), the sequence encoding the packaging signal and structural proteins Spike (S), Envelope (E), Membrane (M), and Nucleocapsid (N) proteins of SARS-CoV-2 Omicron BA.1; or the sequence encoding Spike, Envelope, and Membrane proteins, was inserted into the pSFV3 backbone, and the replicon plasmid vector was subjected to in vitro transcription (IVT). As a result, each band corresponding to the mRNA for each structural protein (S, E, M, and N) of SARS-CoV-2 and the mRNA containing the sequence encoding Spike, Envelope, and Membrane proteins was confirmed. This confirmed that the replicon plasmid vector was converted to mRNA form through IVT.

[0293] As shown in Fig. 5, after the mRNA produced through the IVT was transfected into the BHK-21 cell line, the proteins produced within the cells were confirmed through an immunofluorescence assay, and it was confirmed that the structural proteins of SARS-CoV-2, namely spike, membrane protein, and nucleocapsid protein, were expressed within the cells.

[0294] In addition, the secreted VLPs were purified from the lysates and supernatants of the transfected cells, and Western blot analysis revealed that VLPs were formed in both cases where mRNA encoding each structural protein (S, E, M, and N) of SARS-CoV-2 was transfected and where mRNA containing sequences encoding spike, envelope, and membrane proteins was transfected. In particular, it was confirmed that VLPs were better produced when mRNA containing sequences encoding spike, envelope, and membrane proteins was transfected (Fig. 6).

[0295] Through the above results, it was confirmed that VLPs were stably produced when a vector system containing the structural proteins of SARS-CoV-2 omicron BA.1, including spike, envelope and membrane proteins, and a packaging signal, was transfected.

[0296]

[0297] (2) Confirmation of the stability of the vector system in HEK 293-N cell line

[0298] As shown in Fig. 7, a cell line (HEK 293-N cell line) that stably and constantly expresses the nucleocapsid was constructed, and when the replicon plasmid vector (pSFV3-SEM-PS9) was transfected with the sequence of the structural proteins (S, E, and M) and the packaging signal of SARS-CoV-2 Omicron BA.1 excluding the nucleocapsid instead of the sequence encoding the structural proteins of SFV, it was confirmed whether VLPs were produced.

[0299] As shown in Fig. 8, when the replicon plasmid vector (pSFV3-SEM-PS9) was IVTed, it was confirmed that a band (approximately 13,000 bp) representing the target saRNA was formed.

[0300] As shown in Fig. 9, after the saRNA produced through the IVT was transfected into the HEK 293-N cell line, the proteins produced within the cells were confirmed through an immunofluorescence assay, and it was confirmed that the structural proteins (spike, nucleocapsid, and membrane proteins) of SARS-CoV-2 were expressed within the cells.

[0301] In addition, the secreted VLPs were purified from the lysates and supernatants of the transfected cells, and Western blot analysis was performed to confirm that SARS-CoV-2 VLPs were formed when saRNA containing sequences encoding the structural proteins of SARS-CoV-2, namely spike, envelope, and membrane proteins, was transfected (Fig. 10).

[0302] Through the above results, we confirmed that when the replicon plasmid vector (pSFV3-SEM-PS9) system was stably transfected, the HEK 293-N cell line, which always expresses the nucleocapsid, also stably produced VLPs through the vector system. Accordingly, a VLP mass production cell line (Fig. 11) expressing the SARS-CoV-2 nucleocapsid was constructed to enable repetitive VLP production using the Vero E6 cell line.

[0303]

[0304] <Example 2> Confirmation of the characteristics of VLPs containing saRNA

[0305] To confirm the characteristics of VLPs containing saRNA, the structure and size of VLPs manufactured according to Manufacturing Examples 1 to 3 were measured.

[0306] As shown in Fig. 12, the structure of the VLP containing saRNA including the structural protein, RdRp, and packaging sequence of SARS-CoV-2 was confirmed using transmission electron microscopy (TEM) and showed an amorphous structure. As shown in Fig. 13, the size of the VLP was measured using dynamic light scattering (DLS) and showed that the VLP had a diameter of approximately 56 nm.

[0307] As a result of confirming the amount of VLP produced in the cell line (HEK 293-N) producing the above VLP, 10.6 Log at all locations was observed, as shown in Table 2 below. 10 This indicates that VLPs of more than copies / mL were produced, confirming that a sufficient amount of VLPs for inoculation was stably produced through the vector system.

[0308] Log 10 copies / mLSite 1Site 2Site 3Site 4Site 5Site 612.310.612.512.212.311.9

[0309]

[0310] <Example 3> Confirmation of in vitro cell infectivity of VLP containing saRNA

[0311] To confirm the cell infectivity of VLPs containing saRNA, the infectivity of VLPs manufactured according to Manufacturing Examples 1 to 3 in cells (2D culture) and human bronchial epithelial cells (3D culture) with hACE2 and TMPRSS2 receptors was evaluated using Western blot, immunofluorescence assay, and ISH. In addition, TEM images and Western blots of VLP-infected cells were used to determine whether reinfection occurred after VLP infection.

[0312]

[0313] (1) Confirmation of in vitro cell infectivity of VLP containing saRNA

[0314] As shown in Fig. 14, it was confirmed that VLPs containing saRNA had infectious properties against a cell line (HACE2-293T) expressing hACE2, a host cell receptor used by the SARS-CoV-2 virus to infect the host, and a cell line (A549-HACE2-TMPRSS2) expressing the host protease TMPRSS2 together with hACE2.

[0315] In addition, as shown in Fig. 15, when VLPs were infected in cell lines expressing hACE2 and TMPRSS2, it was confirmed that the spike protein of SARS-CoV-2 was expressed, confirming that the VLPs containing the saRNA have the ability to infect cells.

[0316] As shown in Figures 16 and 17, the infectivity of VLPs in 3D cultured human bronchial epithelial cells was confirmed through an immunofluorescence assay, confirming that the spike protein of SARS-CoV-2 (Figure 16) was expressed in human bronchial epithelial cells. In addition, ISH confirmed that the spike gene of SARS-CoV-2 was expressed in human bronchial epithelial cells, and the ISH results also confirmed that saRNA was distributed within the VLPs (Figure 17).

[0317]

[0318] (2) Confirmation of the possibility of reassembling VLPs containing saRNA

[0319] To determine whether VLPs containing saRNA can reassemble, Western blot was performed on lysates of cells infected with VLPs. As shown in Figure 18, when the cell supernatant was examined 2 days after infection of A549-HACE2-TMPRSS2 cells with VLPs containing saRNA, the nucleocapsid of the VLPs containing saRNA was not detected in the cell supernatant, confirming that the VLPs do not reassemble after cell infection.

[0320] In addition, as shown in Fig. 19, after infecting VERO E6 cell line with VLP, cells were examined using TEM after 30 minutes and 48 hours. As a result, it was confirmed that the cells were infected with VLP after 30 minutes, and after 48 hours, VLP was not observed in the cells, confirming that VLP was not reassembled after a single infection.

[0321] That is, VLPs containing saRNA exhibit high immunogenicity with a single infection because, after infecting a host cell, the saRNA contained within the VLP causes the structural protein of SARS-CoV-2 to be expressed, forming SARS-CoV-2 VLPs without saRNA inside, and since they do not reassemble, the possibility of additional infection is significantly low, thus exhibiting excellent safety.

[0322]

[0323] <Example 4> Confirmation of in vivo mouse infection ability of VLP containing saRNA

[0324] To confirm the infectivity of VLPs containing saRNA in mice, VLPs prepared according to Manufacturing Examples 1 to 3 were administered to mice (hACE2-Tg mice and BALB / c mice) prepared according to Reference Example 4, and the VLP infectivity in mouse lung tissues and the immunogenicity and protective capacity in mice following VLP administration were measured together.

[0325]

[0326] (1) Confirmation of the infectivity of VLPs containing saRNA

[0327] According to Reference Example 5, immunohistochemistry (IHC) and in situ hybridization (ISH) analyses were performed on lung tissues of hACE2-Tg mice nasally administered VLPs containing saRNA to confirm VLP infection.

[0328] As illustrated in Figure 20, the spike gene of SARS-CoV-2 was confirmed to be expressed in the lungs of mice 1 and 2 days after intranasal inoculation with a VLP vaccine containing saRNA. Furthermore, as illustrated in Figure 21, the membrane protein of SARS-CoV-2 was confirmed to be expressed in mouse lung tissue 1 day after vaccination.

[0329] According to the above results, it was confirmed that the VLP vaccine containing saRNA exhibited excellent infectivity against mice.

[0330]

[0331] (2) Confirmation of immunogenicity and protective capacity (BALB / c mice)

[0332] 10 VLP vaccines containing saRNA (sequence encoding the structural protein of Omicron BA.1) 10 After a single intranasal administration (IN) of 1 copy / mice, blood was collected from the mice and 1LD 50 The patient was re-vaccinated with the Wuhan MA virus, and blood samples were collected for 10 days to monitor the progress (Fig. 22).

[0333] 28 days after vaccination with the VLP vaccine containing the saRNA, both α-spike RBD IgG and α-spike trimer IgG increased compared to the control group (PBS) (Fig. 23). In the case of α-spike RBD IgA and α-spike trimer IgA, which exhibit mucosal immunity, it was confirmed that the group that was administered the VLP vaccine containing saRNA intranasally increased significantly compared to the control group and the group that was administered the vaccine intramuscularly (IM). That is, in the case of IgA, which is a mucosal immunity antibody, the antibody titer was significantly higher only when administered intranasally, confirming that nasal administration of the vaccine can induce significantly superior antibody formation (Fig. 24).

[0334] In addition, in the group administered intranasally with the VLP vaccine containing the saRNA, the neutralizing antibody titers against Omicron BA.1 and Wuhan MA were significantly increased compared to the control group and the group administered intramuscularly (IM) with the vaccine (Fig. 25). It was also confirmed that the secretion of INF-γ, which is responsible for innate immunity, was increased (Fig. 26), and the expression of INF-α and INF-β genes was significantly increased (Fig. 27). It was also confirmed that the number of T cells present in the spleen increased compared to the control group (Fig. 28).

[0335] After administering the VLP vaccine containing the saRNA, mice were reinfected with the Wuhan MA virus 28 days later. As a result, the group that received the VLP vaccine containing saRNA intranasally showed a faster recovery in body weight to normal levels compared to the control group and the intramuscular administration group (Fig. 29), a significantly better survival rate (Fig. 30), and a rapid decrease in the virus titer in the lungs of the mice (Fig. 31). In response, the improvement in lung function of the mice was also significantly better than that of the control group (Fig. 32).

[0336] When the above results are summarized, when a VLP vaccine containing saRNA (a sequence encoding the structural protein of Omicron BA.1) was administered intranasally, i.e., when mucosal immunity was induced, mucosal immunity, T cell immunity, innate immunity, and antibody production were significantly superior to when it was administered intramuscularly. In addition, when reinfected with the virus 4 weeks later, it was confirmed that the body weight, lung function, pulmonary viral titer, and survival rate of the mice quickly improved to normal levels through adaptive immunity.

[0337] Accordingly, it was confirmed that the above VLP vaccine can act as an effective vaccine against the SARS-CoV-2 virus and can be utilized as a vaccine that can be administered through mucosal administration.

[0338]

[0339] (3) Confirmation of immunogenicity and protective capacity (hACE2-Tg mice)

[0340] 10 VLP vaccines containing saRNA (sequence encoding structural protein of Omicron BA.1) as in (2) above 10 After a single intranasal (IN) administration of SARS-CoV-2 VLPs to hACE2-Tg mice with receptors at 1LD / mice, blood samples were collected from the mice and 1LD 50 The patient was re-vaccinated with the Wuhan MA virus, and blood samples were collected for 10 days to monitor the progress (Fig. 33).

[0341] Twenty-eight days after vaccination with the VLP vaccine containing the saRNA, both α-spike RBD IgG and α-spike trimer IgG significantly increased compared to the control group (PBS), the intramuscular administration group of the VLP vaccine, and the intranasal administration group of the LNP vaccine (Fig. 34). α-spike RBD IgA and α-spike trimer IgA, which exhibit mucosal immunity, also significantly increased compared to the control group (PBS), the intramuscular administration group of the VLP vaccine, and the intranasal administration group of the LNP vaccine. That is, in the case of IgA, which is a mucosal immunity antibody, the antibody titer was significantly higher only when administered intranasally, confirming that nasal administration of the vaccine can induce significantly superior antibody formation (Fig. 35).

[0342] In addition, in the group administered intranasally the VLP vaccine containing the saRNA, the neutralizing antibody titers against Omicron BA.1, Wuhan MA, and Omicron XBB.1.17 were significantly increased compared to the control group (PBS), the intramuscular administration group of the VLP vaccine, and the intranasal administration group of the LNP vaccine (Fig. 36), and it was confirmed that the secretion of INF-γ, which is responsible for innate immunity, was increased (Fig. 37), and the expression of INF-α and INF-β genes was significantly increased (Fig. 38). In addition, the body weight of the group administered intranasally the VLP vaccine was maintained within the normal weight range without weight loss for up to 10 days after vaccination, confirming that the VLP vaccine safely induces a rapid immune response against the virus without toxicity (Fig. 39).

[0343] After administering the VLP vaccine containing the saRNA, mice were reinfected with the Wuhan MA virus 28 days later. As a result, the group that received the VLP vaccine containing saRNA intranasally showed a rapid recovery in body weight to a normal level compared to the control group (PBS), the intramuscular administration group of the VLP vaccine, and the intranasal administration group of the LNP vaccine (Fig. 40), a significantly superior survival rate (Fig. 41), and a rapid decrease in the titer of the virus present in the lungs and mucosa of the mice (Fig. 42). In response, the effect of improving lung function in the mice was also significantly superior to the control group (PBS), the intramuscular administration group of the VLP vaccine, and the intranasal administration group of the LNP vaccine (Fig. 43).

[0344] When the above results are summarized, when a VLP vaccine containing saRNA (a sequence encoding the structural protein of Omicron BA.1) was administered intranasally, that is, when mucosal immunity was induced, mucosal immunity, T cell immunity, innate immunity, and antibody production were significantly superior compared to when it was administered intramuscularly or when it was administered intranasally as an LNP vaccine. In addition, when reinfected with the virus 4 weeks later, it was confirmed that the body weight, lung function, pulmonary viral titer, and survival rate of the mice were quickly improved to normal levels through adaptive immunity.

[0345] Accordingly, it was confirmed that the VLP vaccine can act as a more effective vaccine against the SARS-CoV-2 virus than the existing LNP type vaccine, and can be used as a vaccine that can induce mucosal immunity as a vaccine that can be administered through the mucosa, which is the infection route of SARS-CoV-2.

[0346]

[0347] <Example 5> Utilization of a vector system for manufacturing VLPs containing saRNA

[0348] In order to construct a vector system for manufacturing VLPs containing saRNA, it was confirmed whether it is possible to insert and utilize a foreign gene other than the sequence encoding the structural protein of Omicron BA.1 into the vector used, whether additional antigens can be co-expressed, and whether the application of chimeric VLPs is possible.

[0349]

[0350] (1) Confirmation of the possibility of foreign gene expression in the vector system for VLP production

[0351] To determine whether non-structural foreign genes of SARS-CoV-2 omicron BA.1 could also be expressed, the sequence of the LacZ protein was subcloned into the pSFV3 backbone (including NSP1 to NSP4, which are non-structural proteins of Semliki Forest Virus) to construct a replicon plasmid, pSFV3-LacZ, which was linearized and the prepared RNA was transfected into BHK-21 and NP-293 cell lines. LacZ expression was confirmed by beta-galactosidase staining on days 1 and 2.

[0352] As shown in Figure 44, it was confirmed that the LacZ protein was expressed in both the BHK-21 cell line and the NP-293 cell line. In other words, it was confirmed that the above VLP production vector system can be utilized by inserting not only a gene expressing a protein derived from SARS-CoV-2 but also a foreign gene (e.g., a gene expressing a protein derived from another virus, etc.) into the VLP production vector.

[0353]

[0354] (2) Confirmation of the possibility of delivering additional antigens other than saRNA

[0355] To confirm the possibility of the above vector system as an additional antigen delivery system, a sequence encoding a luciferase gene and a packaging signal of SARS-CoV-2 Omicron BA.1, which serve as additional antigens, were subcloned into the pSP6-26s plasmid according to Manufacturing Example 1 to construct a pSP6-luciferase vector, and after linearization of the vector together with pSFV3-SEM-PS9 (same as pSFV3-SARS2), the vector was transfected into the NP-293 cell line to confirm the expression of the additional antigen, luciferase (Fig. 45).

[0356] As shown in Figure 46, the expression of the gene expressing the NSP1 protein, the gene expressing the spike protein, and the gene expressing the luciferase was confirmed, and the expression of all three genes was high. Through this, it was confirmed that the gene expressing the luciferase, which was simultaneously transfected as an additional antigen, inhibited the expression of the RNA for VLP production, that is, it did not compete with the RNA for VLP production and was not expressed, and rather, when expressed together with the RNA for VLP production, it showed a higher expression level than when expressed by infecting only the luciferase RNA.

[0357] In addition, VLPs containing additional antigens (luciferase) and saRNA (SARS-CoV-2 BA.1) were vaccinated to BALB / c mice infected with SARS-CoV-2 JIN.1 and uninfected mice, respectively (Fig. 47). As a result, it was confirmed that the additional antigen, luciferase, was expressed in both SARS-CoV-2 JIN.1-infected and uninfected mice (Figs. 47 and 48), which indicates that VLPs containing additional antigens can be vaccinated as a vaccine even for those who have already been infected with the virus or have been vaccinated, and that VLPs containing saRNA (SARS-CoV-2 BA.1) can act as additional antigen delivery vehicles and also have significantly superior antigen delivery ability.

[0358] That is, when a vector containing a sequence encoding an additional antigen is co-transfected with a vector for VLP production, the RdRp included in the RNA expressed by the vector for VLP production recognizes 26s in the RNA sequence expressed by the vector containing the sequence encoding the additional antigen, thereby significantly increasing the expression of the additional antigen without interfering with the expression of saRNA. Therefore, it was confirmed that the vector system for VLP production can also be utilized as a vehicle for delivering an additional antigen.

[0359]

[0360] (3) Confirmation of the feasibility of manufacturing chimeric VLPs

[0361] As shown in Figures 49 and 50, in order to use the vector system as a template applicable to vaccines of other viruses, the possibility of producing chimeric VLPs by changing the receptor binding domain (RBD) of VLPs produced through the vector system was confirmed.

[0362] Specifically, it was confirmed that VLPs containing each altered spike protein were stably formed when the spike protein, i.e., the receptor binding site, of the existing SARS-CoV-2 Omicron BA.1 was changed to Omicron XBB.1 (hereinafter, XBB.1 chimeric VLP; FIG. 49) or MERS-CoV (hereinafter, MERS-CoV chimeric VLP; FIG. 50).

[0363] In addition, when RNA expressing a vector for producing MERS-CoV chimeric VLP was transfected into the VERO E6 cell line, a cell line for mass production of VLPs containing nucleocapsids, it was confirmed that the RBD of MERS-CoV was expressed (Fig. 51), and genes expressing the envelope and membrane proteins of the MERS-CoV chimeric VLP were expressed (Fig. 52), confirming that chimeric VLP can also be stably produced through the VLP mass production cell line.

[0364] That is, even if the sequence portion encoding the structural protein of the virus within the vector system is changed to a sequence encoding the structural protein of another mutant or another virus, VLPs are stably manufactured and mass production is also possible, so it was confirmed that the vector system is useful for using as a vaccine template for various viruses.

[0365]

[0366] (4) Confirmation of immunogenicity and protective capacity of chimeric VLPs

[0367] (4)-1 Confirmation of immunogenicity and protective capacity (XBB.1 chimeric VLP, BALB / c mice)

[0368] To confirm the immunogenicity and protective capacity in mice following administration of the XBB.1 chimeric VLP, the XBB.1 chimeric VLP vaccine was administered intranasally (IN), and then blood was collected from BALB / c mice, which were then re-vaccinated with the Omicron XBB.1 virus. Blood was collected for 10 days to check the progress (Fig. 53).

[0369] 28 days after vaccination with the XBB.1 chimeric VLP vaccine, the neutralizing antibody titers for Omicron BA.1 and Omicron XBB.1 in the group administered the XBB.1 chimeric VLP vaccine intranasally were significantly increased compared to the control group and the group administered the vaccine intramuscularly (IM) (Fig. 54), and it was confirmed that the expression of INF-α and INF-β genes in the group administered the XBB.1 chimeric VLP vaccine intranasally was significantly increased compared to the control group (Fig. 55).

[0370] In addition, after administering the XBB.1 chimeric VLP vaccine, when the mice were reinfected with the Omicron XBB.1 virus 28 days later, it was confirmed that the titer of the virus present in the lungs of the mice in the group administered the XBB.1 chimeric VLP vaccine intranasally also decreased rapidly compared to the control group and the intramuscular administration group (Fig. 56).

[0371] In summary of the above results, when the XBB.1 chimeric VLP vaccine was administered intranasally, i.e., when mucosal immunity was induced, the immune response was significantly superior compared to the control group (PBS) and intramuscular administration, and it was confirmed that even when reinfected with the virus 4 weeks later, the virus titer in the lungs of the mice was quickly improved to a normal level through adaptive immunity.

[0372] Accordingly, it was confirmed that the chimeric VLP vaccine can act as an effective vaccine against the SARS-CoV-2 virus and can be utilized as a vaccine that can be administered through mucosal administration.

[0373]

[0374] (4)-2 Confirmation of immunogenicity and protective capacity (XBB.1 chimeric VLP, hACE2-Tg mice)

[0375] To confirm the immunogenicity and protective capacity in mice following XBB.1 chimeric VLP administration, the XBB.1 chimeric VLP vaccine was administered intranasally (IN), and then blood was collected from hACE2-Tg mice, which were then re-vaccinated with Omicron XBB.1 virus. Blood was collected for 10 days to check the progress (Fig. 57).

[0376] Similar to the experimental results for the above BALB / c mice, 28 days after vaccination with the XBB.1 chimeric VLP vaccine, the neutralizing antibody titers for Omicron BA.1 and Omicron XBB.1 increased in the group administered the XBB.1 chimeric VLP vaccine intranasally compared to the control group and the group administered the vaccine intramuscularly (IM) (Fig. 58), and it was confirmed that the expression of INF-α and INF-β genes in the group administered the XBB.1 chimeric VLP vaccine intranasally increased significantly compared to the control group (Fig. 59).

[0377] In addition, after administering the XBB.1 chimeric VLP vaccine, when the mice were reinfected with the Omicron XBB.1 virus 28 days later, it was confirmed that the group that received the XBB.1 chimeric VLP vaccine intranasally maintained a significantly higher survival rate of mice compared to the control group and the intramuscular administration group, and the body weight also did not decrease (Figs. 60 and 61). In addition, it was confirmed that the titer of the virus present in the lungs of the mice also decreased rapidly (Fig. 62).

[0378] In summary of the above results, when the XBB.1 chimeric VLP vaccine was administered intranasally, i.e., when mucosal immunity was induced, the immune response was significantly superior compared to the control group (PBS) and intramuscular administration. In addition, when reinfected with the virus 4 weeks later, it was confirmed that the virus titer in the lungs was quickly improved to a normal level through adaptive immunity, and a high survival rate was shown through an excellent immune response.

[0379] Accordingly, it was confirmed that the chimeric VLP vaccine can act as an effective vaccine against the SARS-CoV-2 virus and can be utilized as a vaccine that can be administered through mucosal administration.

[0380]

[0381] (4)-3 Confirmation of cell infectivity, immunogenicity, and protective capacity (MERS-CoV chimeric VLP)

[0382] To confirm the presence of cell infectivity of the chimeric VLP, the VERO E6 cell line was inoculated with the MERS-CoV chimeric VLP, and then the expression of the MERS-CoV RBD protein was confirmed.

[0383] As illustrated in Figure 63, when MERS-CoV chimeric VLPs were inoculated into VERO E6 cells, expression of the MERS-CoV RBD protein was confirmed. Thus, it was confirmed that the MERS-CoV chimeric VLPs have cell infectivity.

[0384] In addition, to confirm the immunogenicity and protective effect in mice following MERS-CoV chimeric VLP administration, Hdpp4 mice were blood collected after intranasal (IN) administration of the MERS-CoV chimeric VLP vaccine, and then re-vaccinated with the Omicron MERS-CoV virus. Blood was collected for 10 days to confirm the progress (Fig. 64).

[0385] After vaccination with the MERS-CoV chimeric VLP, α-BA.1 Spike RBD IgG, α-MERS Spike RBD IgG, α-BA.1 Spike Trimer IgG, and α-MERS Spike Trimer IgG all significantly increased compared to the control group (PBS) (Fig. 65). In addition, in the group that received the MERS-CoV chimeric VLP vaccine intranasally, the neutralizing antibody titers against Omicron BA.1 and MERS-CoV significantly increased compared to the control group (Fig. 66). As a result of reinfection with the MERS-CoV virus after administration of the MERS-CoV chimeric VLP vaccine, it was confirmed that the survival rate of mice in the group that received the MERS-CoV chimeric VLP vaccine intranasally was significantly higher than that of the control group, and their body weights also quickly returned to the normal range (Figs. 67 and 68). In addition, it was confirmed that the titer of the virus present in the lungs of the mice also rapidly decreased (Fig. 69).

[0386] When the above results are summarized, it was confirmed that when the MERS-CoV chimeric VLP vaccine was administered intranasally, i.e., when mucosal immunity was induced, the immune response was significantly superior, and even when reinfected with the virus, the viral titer in the lungs was quickly improved to a normal level through adaptive immunity, and a high survival rate was shown through the excellent immune response.

[0387] Accordingly, it was confirmed that the chimeric VLP vaccine can act as an effective vaccine against coronavirus and can be utilized as a vaccine that can be administered through mucosal administration.

Claims

1. A vector containing a gene encoding a nonstructural protein of an alphavirus and a gene encoding a target protein.

2. In claim 1, the alpha virus is Aura virus, Babanki virus, Barmah Forest virus, Bebaru virus, Cabassou virus, Chikungunya virus, Eastern equine encephalitis virus, Everglades virus, Fort Morgan virus, Getah virus, Highlands J virus, Kyzylagach virus, Mayaro virus, Me Tri virus, Middelburg virus, Mosso das Pedras virus, Mucambo virus, Ndumu virus, O'nyong-nyong virus, A vector, wherein at least one virus is selected from the group consisting of Pixuna virus, Rio Negro virus, Ross River virus, Salmon pancreas disease virus, Semliki Forest virus (SFV), Southern elephant seal virus, Tonate virus, Trocara virus, Una virus, Western equine encephalitis virus, and Whataroa virus.

3. A vector according to claim 1, wherein the gene encoding the nonstructural protein of the alpha virus is at least one selected from the group consisting of a gene encoding NSP1 protein, a gene encoding NSP2 protein, a gene encoding NSP3 protein, and a gene encoding NSP4 protein.

4. A vector according to claim 1, wherein the gene encoding the nonstructural protein of the alpha virus comprises a gene encoding RNA-dependent RNA polymerase (RdRp).

5. A vector according to claim 1, wherein the target protein is a target virus-derived protein or a fragment thereof.

6. A vector according to claim 5, wherein the gene encoding the target virus-derived protein or a fragment thereof is at least one selected from the group consisting of a gene encoding the target virus-derived spike protein or a fragment thereof, a gene encoding the target virus-derived envelope protein or a fragment thereof, a gene encoding the target virus-derived membrane protein or a fragment thereof, a gene encoding the target virus-derived packaging signal-related protein or a fragment thereof, and a gene encoding the target virus-derived nucleocapsid protein or a fragment thereof.

7. A vector according to claim 5, wherein the target virus is a virus causing a respiratory disease.

8. A vector according to claim 7, wherein the respiratory disease-causing virus is at least one selected from the group consisting of Coronavirus, Human Respiratory Syncytial Virus, Adenovirus, Influenza Virus, Rhinovirus, Enterovirus, Bocavirus, Parainfluenza Virus, Metapneumovirus, Epstein-Barr Virus, Cytomegalovirus, Herpes Simplex Virus, Measles Virus, and varicella-zoster virus.

9. A method for producing cells for producing virus like particles (VLPs), comprising the step of introducing RNA expressed by the vector of claim 1 into the cells.

10. A method for producing a cell according to claim 9, wherein the step of introducing the RNA into the cell comprises simultaneously introducing RNA expressed by a vector including a gene encoding a packaging signal-related protein derived from a target virus; the alpha virus-derived polynucleotide; and a gene encoding a second target protein.

11. A method for producing a cell according to claim 9, wherein the RNA comprises RNA encoding an RNA-dependent RNA polymerase of an alpha virus and RNA encoding a target protein.

12. In claim 11, the RNA encoding the target protein is at least one selected from the group consisting of RNA encoding a target virus-derived spike protein or a fragment thereof, RNA encoding a target virus-derived envelope protein or a fragment thereof, RNA encoding a target virus-derived membrane protein or a fragment thereof, and RNA encoding a target virus-derived packaging signal-related protein or a fragment thereof. A method for producing a cell, wherein the cell is genetically engineered to express a target virus-derived nucleocapsid protein or a fragment thereof.

13. A method for producing a cell according to claim 9, further comprising a step of linearizing the vector prior to the step of introducing the RNA into the cell.

14. A method for producing a cell according to claim 9, wherein the cell is a microorganism, an animal cell, or a plant cell.

15. A cell for producing virus-like particles manufactured by the method of claim 9.

16. A method for producing a virus-like particle, comprising the step of culturing the cell of claim 15.

17. A virus-like particle manufactured by the method of claim 16.

18. A virus-like particle according to claim 17, wherein the target protein is a target virus-derived spike protein or a fragment thereof, a target virus-derived envelope protein or a fragment thereof, and a target virus-derived membrane protein or a fragment thereof.

19. A virus-like particle according to claim 18, wherein the RNA is packaged with the target virus-derived spike protein or a fragment thereof, the target virus-derived envelope protein or a fragment thereof, and the target virus-derived membrane protein or a fragment thereof.

20. A virus-like particle according to claim 17, further comprising RNA expressed by a vector comprising a gene encoding a packaging signal-related protein derived from a target virus; the alpha virus-derived polynucleotide; and a gene encoding a second target protein.

21. A vaccine composition for preventing or treating respiratory diseases comprising the virus-like particle of claim 17.

22. A vaccine composition according to claim 21, wherein the vaccine composition induces a mucosal immune response.

23. A vaccine composition according to claim 22, wherein the mucosal immune response is expressed by antigen-specific IgA in the mucosa.

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