Virus-like particles containing capsid proteins joined by linkers

Abstract

The purpose of the present invention is to provide a VLP which can have polyvalent antigenic properties and has two capsid proteins present in the same proportion.　A virus-like particle containing an aggregate of a dimer of a first capsid protein and a second capsid protein, wherein the first capsid protein and the second capsid protein are connected to one another by a linker.

Description

[Technical Field]

[0001] The present application relates to a dimer of capsid proteins linked by a linker, a virus-like particle comprising the dimer, an expression vector and a host cell for producing the virus-like particle, and a pharmaceutical composition containing the dimer and / or the virus-like particle. [Background technology]

[0002] When viruses infect other organisms (animals, plants, bacteria, etc.), they cause various symptoms and diseases. In humans, viral infections can be involved in, for example, colds, influenza, bronchitis, pneumonia, gastroenteritis, hepatitis, meningitis, rubella, cancer, and encephalitis. With the recent increase in human traffic, viral infections that were previously limited to specific regions can now spread to other regions. This calls for more effective countermeasures against viral infections.

[0003] Vaccination is widely used as a means of preventing viral infections. Vaccines stimulate the immune function of the subject, thereby enhancing immunity against viruses and preventing infection. To obtain a highly effective vaccine, it is necessary to use an excellent antigen. Therefore, antigens for vaccines are continuously being developed.

[0004] For example, Patent Document 1 discloses a chimeric viral protein 1 (VP1) comprising the shell (S) domain of VP1 from a first norovirus strain and a protruding (P) domain containing at least a portion of the protruding domain of VP1 from a second norovirus strain, as well as a virus-like particle (VLP) comprising the VP1. This document also discloses the preparation of multivalent VLPs by mixing multiple types of VLPs. This technology requires the preparation of a VLP for each antigen (target norovirus strain). Therefore, the greater the number of target antigens, the more time-consuming it is to prepare the multivalent VLP, the more complex quality control is required, the greater the amount of antigen required, and the higher the costs.

[0005] Meanwhile, Patent Document 2 discloses a multivalent VLP composed of antigen proteins derived from two or more norovirus genotypes. Patent Document 3 discloses the preparation of a multivalent VLP by mixing VP1s derived from different norovirus genotypes. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2013-533745 [Patent Document 2] Special Publication No. 2010-505766 [Patent Document 3] Japanese Patent Application Laid-Open No. 2010-539192 Summary of the Invention [Problem to be solved by the invention]

[0007] In the prior art, VLPs with multivalent antigenicity are formed by random self-assembly of multiple different VP1 monomers, which may result in different proportions of different VP1s in the VLP. The present invention aims to provide a VLP that can have multivalent antigenicity, in which two capsid proteins are present in equal proportions. [Means for solving the problem]

[0008] In view of the above, the inventors of the present application focused on linking the first capsid protein and the second capsid protein with a linker, and completed the present invention.

[0009] The present invention provides, but is not limited to, the following: (1) A virus-like particle comprising a dimeric assembly of a first capsid protein and a second capsid protein, the first capsid protein and the second capsid protein being linked by a linker. (2) The virus particle of (1), wherein the linker is a GS linker. (3) A virus-like particle of (2), in which the C-terminus of the first capsid protein and the N-terminus of the second capsid protein are linked via a GS linker. (4) GS linker is (GGGGS) n A virus-like particle according to (2) or (3), wherein n is a linker, and n is an integer of 1 or greater. (5) A virus-like particle according to any one of (1) to (4), wherein the first capsid protein and the second capsid protein are derived from the same or different viruses. (6) A virus-like particle according to any one of (1) to (5), wherein the first capsid protein and the second capsid protein are derived from a virus of the Caliciviridae family. (7) A dimer of capsid proteins in which the first capsid protein and the second capsid protein are linked by a GS linker. (8) A dimer of the capsid protein of (7), wherein the linker is a GS linker. (9) A dimer of the capsid proteins of (8), in which the C-terminus of the first capsid protein and the N-terminus of the second capsid protein are linked via a GS linker. (10) GS linker is (GGGGS) n A dimer of the capsid protein of (8) or (9), wherein n is an integer of 1 or greater. (11) A dimer of any one of the capsid proteins (7) to (10), wherein the first capsid protein and the second capsid protein are derived from the same or different viruses. (12) A dimer of any of the capsid proteins (7) to (11), wherein the first capsid protein and the second capsid protein are derived from a virus of the Caliciviridae family. (13) A polynucleotide comprising a structure in which a polynucleotide encoding a first capsid protein and a polynucleotide encoding a second capsid protein are linked via a polynucleotide encoding a linker. (14) The polynucleotide of (13), wherein the linker is a GS linker. (15) GS linker is (GGGGS)n A polynucleotide according to (14), which is a linker, wherein n is an integer of 1 or greater. (16) The polynucleotide according to any one of (13) to (15), wherein the first capsid protein and the second capsid protein are derived from the same or different viruses. (17) The polynucleotide according to any one of (13) to (16), wherein the first capsid protein and the second capsid protein are derived from a virus of the Caliciviridae family. (18) An expression vector comprising any one of the polynucleotides (13) to (17). (19) A host cell into which the expression vector of (18) has been introduced. (20) A pharmaceutical composition comprising a virus-like particle according to any one of (1) to (6) or a dimer of a capsid protein according to any one of (7) to (12). (21) The pharmaceutical composition according to (20), which is used to induce protective immunity. (22) The pharmaceutical composition according to (20) or (21), which is a vaccine. [Brief explanation of the drawings]

[0010] [Figure 1] Figure 1 shows an example of a conceptual diagram of a dimer of capsid proteins linked by a linker. [Figure 2] Figure 2 shows the results of SDS-PAGE. Marker: molecular weight marker; Washington0207: control sample (VP1 from Washington0207 without a linker); Aomori2-Washington0207: sample containing a dimer of VP1 from Aomori2 and VP1 from Washington0207 linked by a linker; (G4S)4-6: linker with 4 to 6 repeating units. Upper arrow: band around 120 kDa; lower arrow: band around 60 kDa. [Figure 3]Figure 3 shows the results of Western blotting. Marker: molecular weight marker; Washington0207: control sample (Washington0207 VP1 without linker); Aomori2-Washington0207: sample containing a dimer of Aomori2 VP1 and Washington0207 VP1 linked by a linker; (G4S)4-6: linker with 4 to 6 repeat units. Upper arrow: band around 120 kDa; lower arrow: band around 60 kDa. [Figure 4] Figure 4 shows the results of analysis using a transmission electron microscope (TEM). [Figure 5] Figure 5 shows the results of SDS-PAGE. Marker: molecular weight marker; Aomori2: control sample (Aomori2 VP1 without a linker); Aomori2-Washington0207(G4S)5: sample containing a dimer of Aomori2 VP1 and Washington0207 VP1 linked by a linker; (G4S)5: linker with five repeating units. [Figure 6] Figure 6 shows the results of Western blotting. Marker: molecular weight marker; Aomori2: control sample (Aomori2 VP1 without a linker); Aomori2-Washington0207(G4S)5: sample containing a dimer of Aomori2 VP1 and Washington0207 VP1 linked by a linker; (G4S)5: linker with five repeating units. [Figure 7] FIG. 7 shows the results of analysis of linker-linked VLPs by transmission electron microscopy (TEM). [Figure 8] FIG. 8 shows the results of analysis of linker-linked VLPs by size exclusion chromatography (SEC). [Figure 9] FIG. 9 shows the results of analysis of a control sample (Aomori2 VLP) by size exclusion chromatography (SEC). [Figure 10]Figure 10 shows the results of receptor binding inhibitory activity for each VLP when mouse immune serum against GII.4 Aomori2 VLP was used. The BT50 in the figure is the average BT50 obtained using four lots of mouse immune serum. The results obtained using each lot are indicated by open circles (◯). Aomori2: Aomori2 VLP, Washington0207: Washington0207 VLP, Aomori2-Washington0207: Aomori2-Washington0207 linker-linked VLP. *: p<0.05 (Whitney U test). [Figure 11] Figure 11 shows the results of receptor binding inhibitory activity for each VLP when mouse immune serum against GII.4 Washington0207 VLPs was used. The BT50 in the figure is the average BT50 obtained using four lots of mouse immune serum. The results obtained using each lot are indicated by open circles (◯). Aomori2: Aomori2 VLP, Washington0207: Washington0207 VLP, Aomori2-Washington0207: Aomori2-Washington0207 linker-linked VLP. *: p<0.05 (Whitney U test). DETAILED DESCRIPTION OF THE INVENTION

[0011] <Virus-like particles, capsid protein dimers> The present invention provides virus-like particles (hereinafter, sometimes referred to as "VLPs"). Virus-like particles have a structure similar to that of virus particles. However, unlike virus particles, virus-like particles do not contain genetic information (DNA or RNA) and do not have the ability to infect or proliferate. For this reason, virus-like particles can be used as antigens in vaccines against viral infections, etc.

[0012] The virus-like particles of the present invention have a first capsid protein and a second capsid protein. The virus-like particles are formed by the assembly of the first and second capsid proteins. In the present invention, the first capsid protein and the second capsid protein form a dimer, and the virus-like particles are formed by the assembly of multiple dimers. That is, the virus-like particles of the present invention can be said to be an assembly of dimers of the first capsid protein and the second capsid protein. Therefore, the present invention also provides such dimers. The number of dimers that constitute the virus-like particle is not limited. It can be understood that any number of dimers will spontaneously assemble to form a structurally more stable virus-like particle. For example, the virus-like particle can be an assembly of 90 dimers.

[0013] The dimer of the present invention has a structure in which a first capsid protein and a second capsid protein are linked by a linker. The linker may be any type as long as the object of the present invention can be achieved. The structure and repeating units of the linker, as well as the linking mode with the capsid proteins, can be appropriately set depending on the capsid proteins that make up the dimer. For example, the number of repeating units of the linker may be set to 1 or more, preferably 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, the linker may connect the first capsid protein and the second capsid protein via their amino (N) termini, carboxyl (C) termini, side chain amino groups, and / or side chain carboxyl groups. Kishi The linkage can be at any position, such as a carboxyl group, etc. The any position is preferably any position excluding positions that may be included in the epitope region or that may affect the conformation of the epitope region.

[0014] Exemplary linkers include peptide linkers. Peptide linkers can contain any number of amino acids. For example, peptide linkers can be composed of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. Preferably, peptide linkers are composed of 3, 4, 5, 6, or 7 amino acids. A GS linker containing glycine (G) and serine (S) is a preferred example of a peptide linker in the present invention. Linkers with similar flexibility to the GS linker can also be used.

[0015] The method for linking the first capsid protein and the second capsid protein with a linker is not particularly limited. For example, the first capsid protein and the second capsid protein may be linked at any position via a linker by chemical methods. Alternatively, genetic engineering methods can be used. A polynucleotide encoding the first capsid protein and a polynucleotide encoding the second capsid protein can be linked via a polynucleotide encoding a linker, and a dimer in which the first capsid protein and the second capsid protein are linked via a linker can be obtained from the linked polynucleotide using an appropriate expression system. Methods for linking a linker to a protein peptide are known to those skilled in the art. Therefore, the present invention is not limited to the above examples and any known method can be used to link the first capsid protein and the second capsid protein with a linker.

[0016] For example, as a GS linker (G4S) n or (GGGGS) nThe first capsid protein and the second capsid protein can be linked using the GS linker (SEQ ID NO: 1). Here, n is the repeating unit of the linker and is an integer of 1 or greater. Preferably, n is an integer of 2 to 8, more preferably an integer of 4 to 6, and even more preferably 5. The GS linker can link the first capsid protein and the second capsid protein at any position, as long as a virus-like particle can be formed. This any position can preferably be any position excluding positions that may be included in the epitope region or that may affect the three-dimensional structure of the epitope region. For example, the C-terminus of the first capsid protein and the N-terminus of the second capsid protein can be linked by a linker. For example, the N-terminus of the first capsid protein and the C-terminus of the second capsid protein can be linked by a linker.

[0017] The first capsid protein and the second capsid protein may be derived from any virus. The viruses are not particularly limited, and may be, for example, viruses of the same or different families, genera, or species. Generally, capsid proteins derived from viruses of the same genus may have similar three-dimensional structures, so any capsid protein derived from a virus of the same genus can be used. Alternatively, the viruses may be viruses of the same or different genogroups, genotypes, or serotypes. Furthermore, the viruses may be those capable of infecting any host. Examples include bacterial viruses that infect bacteria, plant viruses that infect plants, and animal viruses that infect animals. More specifically, the viruses are viruses of the Caliciviridae family. The Caliciviridae family includes the genera Lagovirus, Norovirus, Hepevirus, Sapovirus, and Nebovirus. Information on the amino acid sequences of viral capsid proteins is available from publicly available sources. Examples of such sources include databases available on the Internet, such as, but not limited to, GenBank, EMBL, and DDBJ (DNA Database of Japan).

[0018] When the first capsid protein and the second capsid protein are both selected from capsid proteins derived from viruses of the same family, genus, or species, the resulting dimer is composed of two identical or different capsid proteins. Thus, a virus-like particle formed by assembly of such dimers will have a monovalent or bivalent antigen of a virus of the same family, genus, or species. Furthermore, when the first capsid protein and the second capsid protein are both selected from capsid proteins derived from viruses of different families, genera, or species, the resulting dimer is composed of two different capsid proteins. Thus, a virus-like particle formed by assembly of such dimers will have a bivalent antigen of a virus of different families, genera, or species.

[0019] Furthermore, when the first capsid protein and the second capsid protein are both selected from capsid proteins derived from viruses of the same genogroup, genotype, or serotype, the resulting dimer is composed of two identical or different capsid proteins. Therefore, a virus-like particle formed by assembly of such dimers will have monovalent or bivalent antigens of viruses of the same genogroup, genotype, or serotype. When the first capsid protein and the second capsid protein are both selected from capsid proteins derived from viruses of different genogroups, genotypes, or serotypes, the resulting dimer is composed of two different capsid proteins. Therefore, a virus-like particle formed by assembly of such dimers will have bivalent antigens of viruses of different genogroups, genotypes, or serotypes.

[0020] From the above explanation, it can be understood that the dimer of the present invention contains the first and second capsid proteins in an equal ratio (1:1). It can also be understood that the virus-like particles of the present invention formed by assembly of the dimers also contain the first and second capsid proteins in an equal ratio (1:1), and may have a regular or uniform structure consisting of the dimers as units. This can be difficult to achieve using conventional techniques that involve mixing capsid protein monomers and allowing them to spontaneously assemble to form virus-like particles. Conventional VLPs are mixtures of VLPs with different capsid protein ratios, and may not have a uniform structure.

[0021] The first and second capsid proteins constituting the virus particles and dimers of the present invention will be described in more detail below, taking as an example the case where these proteins are derived from norovirus. It should be understood that the present invention can also be applied to capsid proteins derived from other viruses.

[0022] Norovirus has 10 genogroups (GI, GII, GIII, GIV, GV, GVI, GVII, GVIII, GIt is known that noroviruses belonging to GI, GII, GIV, GVIII, and GIX infect humans, noroviruses belonging to GIII infect cattle, noroviruses belonging to GV infect mice, noroviruses belonging to GVI infect cats, noroviruses belonging to GVII infect dogs, and noroviruses belonging to GX infect bats. Each genogroup contains genotypes, which are classified or clustered based on the genetic sequence of the norovirus. For example, it is known that various genotypes exist for GI and GII. Currently, there are nine GI genotypes (GI.1-9) and 27 GII genotypes (GII.1-27). Noroviruses include, but are not limited to, Norwalk (M87611), Southampton (L07418), DesertShield 395 (U04469), Chiba 407 (AB042808), Musgrove (AJ277609), BS5 (Hesse) (AF093797), Winchester (AJ277609), B oxer(AF538679), Vancouver730(HQ637267), Hawaii(U07611), Melksham(X81879), SnowMountain(A Y134748), Ibaraki197(LC213885), MK04(DQ456824), Hubei027(MH068811), HuzhouNS17116(MG7633 68), 218001 (MK614154), TV24 (U02030), NS17-A863 (MG892947), NS17-A928 (MG892950), NS17-A1335 (MG892956), Bristol (X76716), Hillingdon (AJ277607), Seacroft (AJ277620), DingHai30 (MH06881) 1), GZ2010-L96(JX989075), NORO_173(MH218642), PA226(MH114014), 15-BA11(MH279838), 016Q01( KY407213), Leeds(AJ277608), Amsterdam(AF195848), VA97207(AY038599), Erfurt546(AF427118),Sw918 (AB074893), Wortley (AJ277618), M7 (AY130761), Tiffin (AY502010), CS-E1 (AY502009), Kawasaki308 (LC037415), OH- QW101(AY823304), OH-QW170(AY823306), Luckenwalde591(EU373815), IF1998(AY675554), Yuri(AB083780), Loreto1847(KT2 90889), Loreto1972(KY225989), Beijing53931(GQ856469), Leon4509(KU306738), Loreto0959(MG495077), Loreto1257(MG49 5079), PNV06929(MG706448), CHDC5191(ACT76139), Camberwell(AF145896), Lordsdale(X86557), Grimsby(AJ004864), Miami Beach(AF414424), Farmington Hills(AY502023), Houston(EU310927), Chiba04-1050(AB220921), Hunter504D(DQ078814), DenHaag89(EF126965), Saga1(AB447456), Aomori2(AB447433) , Yerseke38 (EF126963), Apeldoorn317 (AB445395), Osaka1 (AB541319), OC07138 (AB434770), New Orlens1805 (GU445325), Sydney / NSW0514 (JX459908), Washington0207 (MK754446), and CUHK-NS-2200 (MN400355) (genome accession numbers in parentheses).

[0023] In the present invention, the first and second capsid proteins can be selected from those derived from any norovirus and can be combined in any desired manner. For example, but not limited to, the first and second capsid proteins can be selected from capsid proteins derived from norovirus GI and GII. More specifically, both the first and second capsid proteins can be selected from capsid proteins derived from norovirus GI or GII. Alternatively, one of the first and second capsid proteins can be selected from capsid proteins derived from norovirus GI, and the other can be selected from capsid proteins derived from norovirus GII. Here, the norovirus GI can be any of GI.1 to GI.9, and the norovirus GII can be any of GII.1 to 27. As a further example, a capsid protein derived from norovirus GII.4 can be selected as the first capsid protein, and a capsid protein derived from norovirus GII.17 can be selected as the second capsid protein. Capsid proteins derived from norovirus GII.4 or GII.17 can be selected as the first and second capsid proteins. Of course, the first and second capsid proteins can be interchanged. Because capsid proteins derived from noroviruses can have similar three-dimensional structures across genogroups, genotypes, or strains, any combination of the first and second capsid proteins can be employed, not limited to the combinations of the first and second capsid proteins exemplified above. Here, the capsid protein can include a VP1 capsid protein. Alternatively, the capsid protein can consist of a VP1 capsid protein. Polypeptides (SEQ ID NOS: 10-12) in which the VP1 capsid proteins of norovirus Aomori2 and Washington0207 are linked via a linker (repeating units 4-6) are specific examples of capsid protein dimers of the present invention and components of the virus-like particles of the present invention.

[0024] <Polynucleotides, expression vectors, host cells> The present invention provides a polynucleotide encoding the above-described capsid protein dimer. Because the correspondence between amino acid residues and genetic codons has already been established, it is easy to convert the amino acid sequence of the dimer into the corresponding polynucleotide sequence. The polynucleotide may be either RNA or DNA, but DNA is more convenient in terms of handling and storage.

[0025] Polynucleotides encoding the capsid protein dimers of the present invention can be obtained by biological techniques. For example, RNA encoding viral capsid proteins can be reverse transcribed into cDNA, and the polynucleotides encoding the first and second capsid proteins can be amplified by using the cDNA as a template for gene amplification (reverse transcription PCR). Primers for PCR are designed to amplify a region containing the nucleic acid sequence encoding the first or second capsid protein. That is, primers can be designed to anneal to nucleic acid sequences located upstream and / or downstream of the nucleic acid sequence encoding the first or second capsid protein.

[0026] Sequence information of the polynucleotide encoding the viral capsid protein can also be obtained from publicly available information sources, such as databases publicly available on the Internet (including, but not limited to, GenBank, EMBL, and DDBJ (DNA Database of Japan)).

[0027] Alternatively, polynucleotides encoding the capsid protein dimers of the present invention may be obtained by chemical synthesis. Polynucleotide synthesis can be performed in-house or outsourced. Polynucleotides may be synthesized by any known method. For example, solid-phase synthesis is well known. Those skilled in the art are familiar with the methods and conditions for polynucleotide synthesis.

[0028] The first capsid protein and the second capsid protein can be produced from the polynucleotide encoding the first capsid protein and the polynucleotide encoding the second capsid protein obtained by the above-mentioned method using an appropriate expression system.

[0029] Here, when the first capsid protein and the second capsid protein are produced in separate expression systems, the dimer of the present invention can be obtained by chemically linking the obtained first capsid protein and second capsid protein with a linker, as described above.

[0030] Alternatively, when a peptide linker is used as the linker, a polynucleotide can be prepared in which a polynucleotide encoding a first capsid protein and a polynucleotide encoding a second capsid protein are linked via a polynucleotide encoding the peptide linker. Examples of peptide linkers that can be used include the GS linker (G4S) n or (GGGGS) n and n is a linker (where n is as defined above). Using such a polynucleotide, a dimer in which the first capsid protein and the second capsid protein are linked by a linker can be obtained as the product. This method eliminates the need to separately produce the first capsid protein and the second capsid protein and then chemically link them with a linker.

[0031] The above-mentioned polynucleotides can be subjected to extraction and / or purification procedures as necessary at any stage during their acquisition or use. Those skilled in the art can appropriately select known methods for polynucleotide extraction and / or purification.

[0032] The polynucleotides of SEQ ID NOs: 7 to 9 encode polypeptides in which the VP1 capsid proteins of noroviruses Aomori2 and Washington0207 are linked by a linker (repeat units 4 to 6), and are specific examples of polynucleotides encoding dimers of the capsid proteins of the present invention.

[0033] The polynucleotide encoding the capsid protein dimer of the present invention can be incorporated into an expression vector. Accordingly, the present invention also provides such an expression vector. Examples of expression vectors include pET for expression in E. coli, pAUR for expression in yeast, pIEx-1 for expression in insect cells, and pBApo-CMV for expression in animal cells, but other known vectors can also be used. The polynucleotide can be incorporated into the expression vector by known methods.

[0034] The expression vector of the present invention can be introduced into an appropriate host cell. Accordingly, the present invention provides a host cell into which the expression vector has been introduced. The host cell is not particularly limited, as long as it can produce a capsid protein dimer of the present invention. For example, insect cells (e.g., Sf9, High Five cells, etc.), Escherichia coli, yeast (e.g., Saccharomyces cerevisiae, Saccharomyces pombe, Pichia pastori, etc.), mammalian cells (CHO, HEK, etc.), and any other cells can be used as host cells. By culturing the host cells of the present invention, a capsid protein dimer of the present invention is produced, and the dimer assembles to form the virus-like particle of the present invention. Note that methods for introducing an expression vector into a host cell, culturing the host cell, and extracting, concentrating, or purifying the product are known. These known methods can also be applied to the present invention.

[0035] <Pharmaceutical Composition> The present invention provides a pharmaceutical composition comprising the virus-like particle or capsid protein dimer of the present invention described above.

[0036] The pharmaceutical composition of the present invention may contain any additional component as long as the effects of the invention are achieved. The additional component can be appropriately selected from known components. For example, excipients, diluents, pH adjusters, preservatives, carriers, suspending agents, solubilizers, thickeners, stabilizers, antiseptics, osmotic agents, immunity These include regulators and adjuvants.

[0037] The pharmaceutical composition of the present invention may be administered orally or parenterally, and the intended administration route can be appropriately selected. The pharmaceutical composition of the present invention can be administered via any route, such as intravenous, intraarterial, intramuscular, peritoneal, nasal, transdermal, intradermal, subcutaneous, buccal, sublingual, rectal, oral, ocular, vaginal, pulmonary, or oral.

[0038] The pharmaceutical composition of the present invention may be in the form of, but is not limited to, tablets, capsules, pills, syrup, elixir, emulsion, aerosol, aqueous or non-aqueous injection solution, or powder, granules, or tablets for injection solution (which may be prepared by adding a liquid excipient such as an aqueous or non-aqueous liquid excipient).

[0039] The pharmaceutical compositions of the present invention may treat (prevent, cure, alleviate, or ameliorate) viral infections, virus-induced diseases, or at least one symptom associated with the infection and / or disease. The above explanations apply to viruses in this context. These infections, diseases, or symptoms include, for example, acute gastroenteritis and associated symptoms (at least one of nausea, diarrhea, loose stools, vomiting, nausea, fever, malaise, fatigue, stomach cramps, chills, muscle pain, and headache). However, it should be understood that the present invention may not be limited to these. The prevention, treatment, alleviation, or amelioration may be achieved by, but is not limited to, neutralizing an infectious agent, inhibiting the entry of an infectious agent into cells, inhibiting the replication of an infectious agent, protecting host cells from infection or destruction, or stimulating antibody production.

[0040] The pharmaceutical composition of the present invention can be administered to a subject in an amount effective to prevent, treat, alleviate, or ameliorate a viral infection, a virus-induced disease, or at least one symptom associated with the infection and / or disease. The subject may be any subject that can be infected with a virus, and may be, for example, a mammal (human, pig, cow, rodent, dog, cat, etc.). The effective amount can be determined appropriately taking into account the subject to which the composition is applied, the administration route, the administration form, etc.

[0041] Furthermore, the pharmaceutical compositions of the present invention can induce protective immunity against viruses, thereby being useful for preventing, treating, alleviating, or ameliorating viral infections, virus-induced diseases, or at least one symptom associated with such infections and / or diseases. Here, the induction of "protective immunity" against a virus means eliciting immunity or an immune response against an infectious agent (a virus, a substance derived therefrom, or a substance produced therefrom). The protective immune response may be due to either a humoral immune response or a cell-mediated immune response. Induction of protective immunity against a virus can result in a higher antibody titer against the virus in a subject compared to before the induction. Antibody titers can be measured by known methods. Therefore, the pharmaceutical compositions of the present invention can be used as vaccine compositions.

[0042] The pharmaceutical composition of the present invention can be used, for example, to treat infections, diseases, and symptoms associated with norovirus. Norovirus has been described above. The norovirus targeted by the pharmaceutical composition of the present invention is not limited and may belong to any genogroup or genotype. However, it should be understood that the target norovirus is determined by the origin of the capsid protein that constitutes the virus-like particle or dimer contained in the pharmaceutical composition of the present invention. For example, but not limited to, the virus-like particle or capsid protein dimer contained in the pharmaceutical composition may be: When the pharmaceutical composition contains a capsid protein derived from norovirus GI, the target of the pharmaceutical composition is at least norovirus GI; When the pharmaceutical composition contains a capsid protein derived from norovirus GII, the target of the pharmaceutical composition is at least norovirus GII; When the pharmaceutical composition contains capsid proteins derived from norovirus GI and GII, the target of the pharmaceutical composition is at least norovirus GI and GII.

[0043] As described above, the virus-like particles of the present invention and the capsid protein dimers of the present invention may have a regular or uniform structure containing the first and second capsid proteins in an equal ratio (1:1). This characteristic may be beneficial in terms of quality control of pharmaceutical compositions. Furthermore, when the virus-like particles of the present invention or the capsid protein dimers of the present invention have bivalent or polyvalent antigenicity, this may be beneficial in efficiently producing pharmaceutical compositions with polyvalent antigenicity. It may also be beneficial in that the amount of protein added can be reduced. [Example]

[0044] The present invention will be described in more detail by the following examples, which are provided for the purpose of better understanding the invention and are not intended to limit the scope of the invention.

[0045] [Example 1] Preparation of recombinant baculovirus The VP1 genes from the following norovirus strains were used: (1) VP1 gene (SEQ ID NO: 2) derived from Norovirus Hu / GII-4 / Aomori2 / 2006 / JP (Accession number: AB447433) (hereinafter referred to as "Aomori2"). (2) VP1 gene (SEQ ID NO: 3) derived from Norovirus GII isolate Hu / US / 2018 / GII.P16-GII.4 Sydney / Washington0207 (Accession number: MK754446) (hereinafter referred to as “Washington0207”).

[0046] A G4S linker was used as the linker, with repeating units of 4 ((G4S)4), 5 ((G4S)5), and 6 ((G4S)6). The gene encoding (G4S)4 (SEQ ID NO: 4), the gene encoding (G4S)5 (SEQ ID NO: 5), and the gene encoding (G4S)6 (SEQ ID NO: 6) were obtained.

[0047] DNAs in which two different VP1 genes were linked with a linker were obtained by gene synthesis. The following DNAs were obtained: DNA in which two VP1 genes were linked with a gene encoding a (G4S)4 linker (SEQ ID NO: 7); DNA in which two VP1 genes were linked with a gene encoding a (G4S)5 linker (SEQ ID NO: 8); and DNA in which two VP1 genes were linked with a gene encoding a (G4S)6 linker (SEQ ID NO: 9).

[0048] These DNAs were then transferred to the transfer vectors (pFastBac TM The vector was then inserted into Escherichia coli (MAX Efficiency) carrying baculovirus genomic DNA. TM The vector was introduced into DH10Bac Competent Cells (Thermo Fisher Scientific, Cat. No. 10361012), and the target DNA was integrated into the baculovirus genomic DNA by homologous recombination. Baculovirus genomic DNA was extracted and purified from E. coli (QIAprep Spin Miniprep Kit, QIAGEN, Cat. No. 27106) and introduced into Sf9 cells (Lipofectamine). TM LTX Reagent with PLUS TM After culturing Sf9 cells at 26-28°C for 7 days, the recombinant baculovirus was collected from the culture supernatant and stored at -80°C.

[0049] [Example 2] Preparation of linker-linked VLPs The recombinant baculovirus prepared in Example 1 was inoculated into High Five cells at an appropriate MOI (Multiplicity of Infection). High Five cells were cultured at 26 to 28°C to produce linker-linked VLPs. Four to seven days after infection, the culture medium was collected and centrifuged at 10,000 × g for 60 minutes to separate the culture supernatant and cell pellet. The cell pellet was disrupted using an ultrasonicator and centrifuged at 15,000 × g for 10 minutes to collect the supernatant. The culture supernatant and the supernatant after cell pellet disruption were subjected to density gradient centrifugation using cesium chloride to obtain linker-linked VLPs.

[0050] Example 3: Characterization of VLPs The expression of a linker-linked VP1 protein dimer, a component of the linker-linked VLP, was confirmed by SDS-PAGE and Western blotting.

[0051] SDS-PAGE analysis was performed as follows: 27 μl of the sample prepared in Example 2 was mixed with 9 μl of sample buffer (4× Laemmli Sample Buffer, Bio-Rad Laboratories, Cat. No. #161-0747) (containing DTT) and heated at 95°C for 5 minutes. 12 μl of the heated solution was applied to one lane of an SDS-PAGE gel and electrophoresed at 200 V for 30 minutes. After electrophoresis, the gel was stained with Coomassie (Bio-Safe Coomassie Stain, Bio-Rad Laboratories, Cat. No. #1610787) and then destained with DW for 20 minutes three times.

[0052] Western blotting analysis was performed as follows. Using a transfer device, the proteins on the gel after SDS-PAGE were transferred to a PVDF membrane (200 V, 30 minutes). After immersion in blocking buffer, VP1 of the GII.4 genotype was detected using a 10,000-fold diluted GII.4 genotype detection antibody (anti-VLP antibody of Norovirus Hu / GII.4 / Sydney / NSW0514 / 2012 / AU (JX459908)).

[0053] Each gel image was captured using a gel imager, and the resulting band intensities were then quantified by an analytical panel to calculate the purity of the VLPs.

[0054] The results of SDS-PAGE analysis are shown in Figure 2. In the sample obtained using the recombinant baculovirus prepared in Example 1, a band was detected around 120 kDa (lane "Aomori2-Washington0207" in Figure 2). No band was detected around 120 kDa in the control sample (lane "Washington0207" in Figure 2). A band thought to correspond to the VP1 monomer protein was detected around 60 kDa, suggesting that the 120 kDa band is a protein formed by linking two VP1s (VP1 derived from Aomori2 and VP1 derived from Washington0207) with a linker. Furthermore, a band around 120 kDa was observed regardless of the length of linker used.

[0055] The results of Western blotting are shown in Figure 3. The band detected around 60 kDa was confirmed to correspond to the VP1 monomer. The band was detected at the same position regardless of whether the VP1 was derived from Washington0207 or Aomori2. The band detected around 120 kDa was confirmed to be a protein consisting of two VP1s linked by a linker (lane "Aomori2-Washington0207" in Figure 3). No band was detected around 120 kDa in the control sample (lane "Washington0207" in Figure 3). Furthermore, regardless of the length of the linker used, expression of the protein consisting of two VP1s linked by a linker was confirmed, indicating that expression of the linked protein is independent of the length of the linker.

[0056] [Example 4] Analysis by transmission electron microscope (TEM) The structural characteristics of the linker-linked VP1 dimer protein in the sample prepared in Example 2 were analyzed using TEM.

[0057] TEM analysis was performed as follows: After diluting the sample, it was dropped onto a copper mesh, stained with phosphotungstic acid staining solution, and observed under an electron microscope.

[0058] As a representative example, the results of analysis of a VP1 dimer protein linked by a (G4S)5 linker are shown (Figure 4). This figure confirms that the linker-linked VP1 dimer forms particles (VLPs). These results suggest that the VLP is a chimeric VLP with polyvalent antigenicity against two norovirus strains (Aomori2 and Washington0207). It is expected that two linker-linked VP1 dimer proteins will also form VLPs when other linkers are used.

[0059] [Example 5] Analysis by size exclusion chromatography (SEC) The structural characteristics of the linker-linked VP1 dimer proteins in the samples prepared in Example 2 were analyzed using SEC.

[0060] The SEC conditions were as follows: 50 μl of the purified linker-linked VP1 dimer protein sample was injected. The flow rate was 0.8 ml / min, and a TSKgel G6000PWXL column (Tosoh Bioscience, Cat. No. 0008024) was used. Phosphate buffer (pH 7.4) was used as the sample dilution and mobile phase.

[0061] SEC analysis also confirmed that the linker-linked VP1 dimer protein formed particles.

[0062] [Example 6] Re-preparation and characterization of linker-linked VLPs The recombinant baculovirus prepared in Example 1 was inoculated at an appropriate MOI (multiplicity of infection) into High Five cells cultured at 26-28°C. Four to seven days after inoculation, the culture medium was collected and centrifuged at 10,000 × g for 20 minutes to recover the cell pellet. The cell pellet was disrupted using an ultrasonic disrupter and centrifuged at 15,000 × g for 10 minutes to recover the supernatant. The recovered supernatant was subjected to sucrose cushion centrifugation (40 wt% sucrose solution) to recover a pellet containing linker-linked VLPs from which low-molecular-weight impurities had been removed. This pellet was suspended in PBS and subjected to cesium chloride density gradient centrifugation to obtain purified linker-linked VLPs.

[0063] The resulting linker-linked VLPs were characterized by SDS-PAGE and Western blotting in the same manner as in Example 3, and particle formation was confirmed by electron microscopy (TEM) and size exclusion chromatography (SEC) in the same manner as in Example 4. Furthermore, particle size was evaluated by multi-angle light scattering (MALS).

[0064] SEC was performed under the same conditions as in Example 5, except that 20 μl of sample was injected. The fractions of the peaks separated and detected by SEC were subjected to MALS, and the particle diameter was measured at dn / dc=0.185.

[0065] The results of SDS-PAGE analysis are shown in Figure 5. A single band was detected at approximately 120 kDa for the newly prepared linker-linked VLPs (Figure 5, lane "Aomori2-Washington0207(G4S)5"). In contrast, no band was detected at approximately 120 kDa for the control sample (Figure 5, lane "Aomori2"), and a band likely corresponding to the VP1 monomer protein was detected at approximately 60 kDa. Furthermore, Western blotting results (Figure 6) indicated that the band at approximately 120 kDa for the linker-linked VLPs was detected with an antibody specific for VP1 of GII.4. Therefore, this band was considered to correspond to the target protein, consisting of two types of VP1 (VP1 derived from Aomori2 and Washington0207) linked by a G4S linker. These results confirmed that the preparation of linker-linked VLPs was reproducible.

[0066] Furthermore, the purity of the linker-linked VLPs prepared in this example was higher than that of those prepared in Example 2. This was thought to be partly due to the addition of a sucrose cushion treatment during the purification procedure in this example.

[0067] The structure of the linker-linked VLP prepared in this example was analyzed using TEM. The resulting electron microscope image ( FIG. 7 ) confirmed that the linker-linked VLP formed particles, as in Example 4. SEC analysis revealed that the retention time of the linker-linked VLP ( FIG. 8 ) was almost identical to that of the control sample ( FIG. 9 : Aomori2 VLP). Furthermore, the radius of gyration (rw) measured by MALS was 32.9 nm for the linker-linked VLP and 23.6 nm for the control sample (Aomori2 VLP). These results confirmed that the linker-linked VLP had a particle size roughly equivalent to that of a VLP formed from a single VP1.

[0068] [Example 7] Confirmation of antigenicity of linker-linked VLPs The antigenicity of the linker-linked VLPs was evaluated using a receptor binding inhibition test using porcine gastric mucin (PGM). The test was performed as follows, with reference to Haynes, J et al., Viruses 2019;11(5):392.

[0069] 100 μl of PGM solution (1.0 μg / mL, PBS) prepared using commercially available PGM (catalog no. M1778, Sigma-Aldrich) was added to each well of the well plate and allowed to stand at 25°C for 2 hours. The solution was then removed, and 300 μl of washing buffer (PBS containing 0.05% Tween 20) was added to wash each well (this washing procedure was repeated three times). 200 μl of 5% skim milk-PBS solution was added to each well and allowed to stand at 4°C overnight.

[0070] 200 μl of each solution (0.025 μg / ml) containing three types of VLPs (Aomori2 VLP, Washington0207 VLP, and Aomori2-Washington0207 linker-linked VLP (all prepared according to the method of Example 2)) was mixed with 200 μl of mouse immune serum solution serially diluted in two-fold increments from 40-fold to 20,480-fold, and allowed to stand at 25°C for one hour. This mixture was used as a VLP-mouse immune serum mixture. Mouse immune serum was prepared from blood collected after two doses of each VLP (Aomori2 VLP and Washington0207 VLP) administered to four mice at 100 μg / head (four lots for each VLP).

[0071] After incubating overnight at 4°C, each well of the well plate was washed three times with washing buffer, and 100 μl of the VLP-mouse immune serum mixture was added to each well and incubated for one hour at 25°C. The plate was then washed three times with washing buffer, and 100 μl of primary antibody solution (prepared in-house) (dissolved in PBS containing 0.05% Tween 20) was added to each well and incubated for one hour at 25°C. The primary antibody solution was prepared from blood collected after administering each VLP (Aomori2 VLP and Washington0207 VLP) to rabbits twice at 1 mg / head. The plate was washed three times with washing buffer, and 100 μl of secondary antibody solution (Anti-Rabbit IgG Antibody, HRP conjugated (Catalog #65-6120, Invitrogen)) (dissolved in PBS containing 0.05% Tween 20) was added to each well and allowed to stand at 25°C for one hour. The plate was washed three times with washing buffer, and 100 μl of TMB (3,3',5,5'-tetramethylbenzidine) solution was added to each well. The plate was then allowed to stand at 25°C for 30 minutes in the dark. The reaction was terminated by adding 100 μl of stop solution. The optical density (OD) of the solution at 450 nm after the reaction was stopped was measured.

[0072] The absorbance of the well to which a mixture of VLP and PBS containing 0.05% Tween 20 and 5% skim milk (without mouse immune serum) was added was defined as OD [without serum], the absorbance of the well to which no VLP-mouse immune serum mixture was added was defined as blank, and the absorbance of the well to which the VLP-mouse immune serum mixture was added was defined as OD [with serum]. The binding inhibitory activity was calculated using the following formula. Binding inhibitory activity (%) = 100-[(OD[serum present]-blank) / (OD[serum absent]-blank)]×100

[0073] The presence or absence of receptor binding inhibitory activity was determined based on the blocking titer (BT)50 value. BT50 is the maximum serum dilution at which binding inhibitory activity exceeds 50%. Statistical analysis was performed using the Mann-Whitney rank sum test on the BT50 values ​​obtained for each of the three VLPs (Aomori2 VLP, Washington0207 VLP, and Aomori2-Washington0207 linker-linked VLP). BT50 values ​​were compared between each VLP, and statistical significance was used to determine whether the linker-linked VLPs possessed two types of antigenicity.

[0074] The results of the receptor binding inhibition test are shown in Figures 10 and 11. Figure 10 shows the results of receptor binding inhibitory activity for each VLP (Aomori2 VLP, Washington0207 VLP, and Aomori2-Washington0207 linker-linked VLP) when mouse immune serum (four lots) against Aomori2 VLP was used. Figure 11 shows the results of receptor binding inhibitory activity for each VLP when mouse immune serum (four lots) against Washington0207 VLP was used.

[0075] As shown in Figure 10, the mouse immune serum against Aomori2 VLPs had significantly lower receptor binding inhibitory activity than Washington0207 VLPs. On the other hand, it was confirmed that the mouse immune serum had the same level of receptor binding inhibitory activity against linker-linked VLPs containing Aomori2 VP1 as that of Aomori2 VLPs.

[0076] Furthermore, as shown in Figure 11, mouse immune serum against Washington0207 VLP also showed slight receptor binding inhibitory activity against Aomori2 VLP, but this was significantly lower than that of Washington0207 VLP or linker-linked VLP, and the receptor binding inhibitory activity of Washington0207 VLP and linker-linked VLP was comparable.

[0077] Therefore, the linker-linked VLP of the present invention is a chimeric VLP formed from two norovirus VP1s linked via a linker, and it was considered that the chimeric VLP is a multivalent antigenic particle having the antigenicity of two types of VP1.

Claims

1. comprising a dimeric assembly of a first capsid protein and a second capsid protein; the C-terminus of the first capsid protein and the N-terminus of the second capsid protein are linked by a GS linker; A virus-like particle, the first capsid protein is a VP1 capsid protein derived from a Norovirus; the second capsid protein is a VP1 capsid protein from a Norovirus; Virus-like particles.

2. The GS linker is (GGGGS) ｎ The virus-like particle of claim 1 , wherein n is a linker, and n is an integer of 1 or greater.

3. A virus-like particle described in claim 2, wherein n is an integer from 2 to 8.

4. The virus-like particle of claim 1 , wherein the first capsid protein and the second capsid protein are derived from noroviruses belonging to the same or different genogroups.

5. A virus-like particle as described in claim 1, comprising the first and second capsid proteins in equal proportions (1:1).

6. A dimer of capsid proteins, wherein a first capsid protein and a second capsid protein are linked by a GS linker, the C-terminus of the first capsid protein and the N-terminus of the second capsid protein are linked by a GS linker; the first capsid protein is a VP1 capsid protein derived from a Norovirus; the second capsid protein is a VP1 capsid protein from a Norovirus; Capsid protein dimer.

7. The GS linker is (GGGGS) ｎ A capsid protein dimer according to claim 6, wherein n is a linker, and n is an integer of 1 or greater.

8. A dimer of the capsid protein described in claim 7, wherein n is an integer from 2 to 8.

9. The capsid protein dimer of claim 6, wherein the first capsid protein and the second capsid protein are derived from noroviruses belonging to the same or different genetic groups.

10. A dimer of the capsid protein described in claim 6, comprising the first and second capsid proteins in equal proportions (1:1).

11. A polynucleotide comprising a structure in which a polynucleotide encoding a first capsid protein and a polynucleotide encoding a second capsid protein are linked via a polynucleotide encoding a GS linker, the first capsid protein is a VP1 capsid protein derived from a Norovirus; the second capsid protein is a VP1 capsid protein from a Norovirus; Polynucleotide.

12. The GS linker is (GGGGS) ｎ 12. The polynucleotide of claim 11, wherein n is a linker, and n is an integer of 1 or greater.

13. The polynucleotide of claim 12, wherein n is an integer from 2 to 8.

14. The polynucleotide of claim 11 , wherein the first capsid protein and the second capsid protein are derived from Noroviruses belonging to the same or different genogroups.

15. A polynucleotide described in claim 11, comprising the first and second capsid proteins in equal proportions (1:1).

16. An expression vector comprising the polynucleotide according to any one of claims 11 to 15.

17. A host cell into which the expression vector of claim 16 has been introduced.

18. A pharmaceutical composition comprising the virus-like particle according to any one of claims 1 to 5 or the capsid protein dimer according to any one of claims 6 to 10.

19. 19. The pharmaceutical composition of claim 18 for use in inducing protective immunity.

20. 19. The pharmaceutical composition of claim 18, which is a vaccine.

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