Stabilized VLP vaccines

Abstract

To provide a VLP vaccine derived from the genus Enterovirus which is safe and has good stability.SOLUTION: The VLP vaccine of the present invention is highly safe because it uses VLPs having no viral internal gene as an antigen, and has good stability because it is fixed with formaldehyde in a high salt concentration.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a stabilized VLP vaccine used for preventing diseases caused by Enteroviruses, a method for producing the same, and a method for stabilizing the VLP vaccine. [Background technology]

[0002] Hand, foot, and mouth disease (HFMD) is an acute viral infection whose main symptom is a vesicular rash that appears on the oral mucosa, hands, and feet. The main causative viruses of HFMD are Enteroviruses, such as Coxsackie A16 (CVA16), CVA10, CVA6, and Enterovirus 71 (EV71). Hand, foot, and mouth disease generally has a good prognosis, but it can sometimes be complicated by acute meningitis and, although rare, can cause acute encephalitis. In particular, EV71 is known to have a higher incidence of central nervous system complications than other viruses. Several inactivated enterovirus 71 (EV71) vaccines have been approved for hand, foot, and mouth disease in the People's Republic of China, but all are inactivated whole particle vaccines against genotype C4. Furthermore, a phase 3 clinical trial of a whole particle vaccine, EV71vac, against genotype B4 is currently underway in Vietnam (Non-Patent Document 1), but no hand, foot, and mouth disease vaccines have been approved in Japan. In recent years, virus-like particles (VLP) vaccines, which use VLPs composed only of the viral shell (capsid) as vaccine antigens, have been approved as vaccines for hepatitis B virus and human papillomavirus (Nikkei Shimbun, July 27, 2020, "DNA, mRNA, Vector... What are the Differences Between the Various Vaccines?"). On the other hand, VLP vaccines derived from the genus Enterovirus are still in the preclinical stage. However, Patent Document 1 describes that by expressing the viral structural protein P1 and nonstructural protein 3CD of enterovirus, a VLP vaccine of EV71 in the form of a complete particle composed of VP1 to VP4 was obtained, and that the vaccine showed high immunogenicity. Furthermore, Patent Document 2 describes that EV71 VLPs that can be used as vaccines can be expressed using a CHO cell line as a host cell. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] WO2019 / 163781 [Patent Document 2] WO2020 / 067027 [Non-patent literature]

[0004] [Non-Patent Document 1] The Lancet VOLUME 399, ISSUE 10336, P1708-1717, APRIL 30, 2022 Summary of the Invention

[0005] In light of the above-mentioned circumstances, there is a need for a stable Enterovirus-derived virus-like particle (VLP) vaccine that uses VLPs derived from the Enterovirus genus, such as CVA16 and EV71, as vaccine antigens.

[0006] That is, the present invention provides VLP vaccines derived from Enterovirus genus, in the following embodiments. One embodiment of the present invention is shown below. (A-1) A vaccine comprising stabilized virus-like particles derived from the genus Enterovirus. (A-2) The vaccine according to (A-1), wherein the salt concentration of the vaccine is 200 mM or less. (A-3) The vaccine according to (A-1) or (A-2), wherein the Enterovirus is Enterovirus 71 and / or Coxsackievirus A. (A-4) The vaccine according to any one of (A-1) to (A-3), wherein the virus-like particles have been treated with formaldehyde in a buffer solution containing a salt. (A-5) The vaccine according to (A-4), wherein the salt is selected from acidic, neutral, and basic normal salts. (A-6) The vaccine according to (A-4) or (A-5), wherein the buffer solution contains the salt at a concentration of 500 mM to 3 M. (A-7) The vaccine according to any one of (A-4) to (A-6), wherein the formaldehyde is present at a concentration of 0.01% to 0.2%. (A-8) The vaccine according to any one of (A-4) to (A-7), wherein the treatment is carried out for a period of 1 to 3 weeks. (A-9) The vaccine according to any one of (A-4) to (A-8), wherein the treatment is carried out at a temperature between 4°C and 37°C. (A-10) The vaccine according to any one of (A-1) to (A-9), which is a vaccine for hand, foot and mouth disease.

[0007] Further, other aspects of the present invention are described below. (B-1) A vaccine comprising stabilized virus-like particles derived from the genus Enterovirus, the virus-like particles having a fixed particle structure. (B-2) The vaccine described in (B-1), wherein the stabilization is performed using a buffer containing salt and the fixation is performed using formaldehyde. (B-3) The vaccine according to (B-2), wherein the salt is selected from acidic, neutral, and basic normal salts. (B-4) The vaccine according to (B-2) or (B-3), wherein the salt is contained in the buffer solution at a concentration of 500 mM to 3 M. (B-4) The vaccine according to (B-2), wherein the formaldehyde is present at a concentration of 0.01% to 0.2%. (B-5) The vaccine according to any one of (B-1) to (B-4), wherein the Enterovirus is Enterovirus 71 and / or Coxsackievirus A. (B-6) The vaccine according to any one of (B-1) to (B-5), which is a vaccine for hand, foot and mouth disease.

[0008] Further aspects of the invention are set out below. (C-1) A vaccine comprising stabilized virus-like particles derived from the genus Enterovirus, wherein the Tm value of the main peak of the virus-like particles in differential scanning calorimetry is at least 3°C ​​higher than that of unstabilized virus-like particles. (C-2) The vaccine according to (C-1), wherein the stabilization is performed by a buffer containing a salt. (C-3) The vaccine according to (C-2), wherein the salt is selected from acidic, neutral, and basic normal salts. (C-4) The vaccine according to (C-2) or (C-3), wherein the salt is contained in the buffer solution at a concentration of 500 mM to 3 M. (C-5) The vaccine according to any one of (C-2) to (C-4), wherein the stabilized virus-like particles derived from the genus Enterovirus are further immobilized. (C-6) The vaccine according to (C-5), wherein the fixation is performed using formaldehyde at a concentration of 0.01% to 0.2%. (C-7) The vaccine according to any one of (C-1) to (C-6), wherein the Enterovirus is Enterovirus 71 and / or Coxsackievirus A. (C-8) A vaccine comprising stabilized virus-like particles derived from the genus Enterovirus, wherein at least 90% of the virus-like particles are in native form as measured after overnight incubation at 37°C. (C-9) The vaccine described in (C-8), wherein the Enterovirus is Enterovirus 71 and / or Coxsackievirus A. (C-10) The vaccine described in (C-8), wherein the measurement is selected from the group consisting of absorbance measurement, differential scanning calorimetry (DSC), enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunofluorescence (IF). (C-11) The vaccine according to any one of (C-1) to (B-10), which is a vaccine for hand, foot and mouth disease.

[0009] Further aspects of the present invention are set forth below. (D-1) 1. A method for producing a vaccine comprising stabilized virus-like particles derived from an Enterovirus, said method comprising treating said virus-like particles with formaldehyde in a buffer comprising a salt. (D-2) The method according to (D-1), further comprising a treatment to reduce the salt concentration. (D-3) The method according to (D-1) or (D-2), wherein the salt is selected from acidic, neutral, and basic normal salts. (D-4) The method according to (D-1) or (D-2), wherein the buffer solution contains the salt at a concentration of 500 mM to 3 M. (D-5) The method according to (D-1) or (D-2), wherein the formaldehyde has a concentration of 0.01% to 0.2%. (D-6) The method according to (D-1) or (D-2), wherein the treatment is carried out for a period of 1 to 3 weeks. (D-7) The method according to (D-1) or (D-2), wherein the treatment is carried out at a temperature between 4°C and 37°C. (D-8) The method according to any one of (D-1) to (D-7), wherein the vaccine is a vaccine for hand, foot and mouth disease.

[0010] Further, still another aspect of the present invention is described below. (E-1) 1. A method for stabilizing virus-like particles derived from the genus Enterovirus, said method comprising treating said virus-like particles with formaldehyde in a buffer containing a salt. (E-2) The method according to (E-1), further comprising a treatment to reduce the salt concentration. (E-3) The method according to (E-1) or (E-2), wherein the salt is selected from acidic, neutral, and basic normal salts. (E-4) The method according to (E-1) or (E-2), wherein the buffer solution contains the salt at a concentration of 500 mM to 3 M. (E-5) The method according to (E-1) or (E-2), wherein the formaldehyde has a concentration of 0.01% to 0.2%. (E-6) The method according to (E-1) or (E-2), wherein the treatment is carried out for a period of 1 to 3 weeks. (E-7) The method according to (E-1) or (E-2), wherein the treatment is carried out at a temperature between 4°C and 37°C. (E-8) The method according to any one of (E-1) to (E-7), wherein the Enterovirus is a virus that causes hand, foot, and mouth disease. [Brief explanation of the drawings]

[0011] [Figure 1] This graph shows the change in concentration of native (top row), denatured (middle row), and (native + denatured) (bottom row) CVA16 VLPs with or without heat treatment, measured using three types of neutralizing antibodies. [Figure 2] Graphs showing the amount of native (left), (native + denatured) (middle), and denatured (right) CVA16 VLP and CVA16 VLPs measured using three types of neutralizing antibodies as a function of temperature. [Figure 3]Graph showing the amount of CVA16 VLP (native + denatured) (top row), native (middle row), and denatured (bottom row) for each incubation period at 4°C (left), 25°C (middle row), and 37°C (right) for each concentration of formaldehyde (FA), measured using three types of neutralizing antibodies. [Figure 4] (Left) Graph showing the amount of CVA16 VLP (native + denatured) (top), native (middle), and denatured (bottom) after overnight incubation at 4°C, 25°C, and 37°C; (Right) Graph showing the same measurements for untreated inactivated virus and untreated VLP as controls. [Figure 5] Graph showing the changes in the amount of undenatured (left) and denatured (right) CVA16 VLPs at various NaCl concentrations using phosphate buffer (top row) and HEPES (bottom row). [Figure 6] Graphs showing the changes in the amount of native (left), (native + denatured) (middle), and denatured (right) VLPs of CVA16 with each salt (NaCl, KCl, ammonium sulfate). [Figure 7] Graph showing the change in the amount of native (left), (native + denatured) (middle), and denatured (right) CVA16 VLP after incubation of formaldehyde-immobilized and non-immobilized VLPs overnight at 37°C in a buffer containing 170 mM salt. [Figure 8] Graph showing the change in the amount of CVA16 VLP (native + denatured) (top row), native (middle row), and denatured (bottom row) at each fixation period using 0.01% to 0.05% formaldehyde. [Figure 9] Graph showing the change in the amount of CVA16 VLP (native + denatured) (top row), native (middle row), and denatured (bottom row) at each fixation period using 0.05% to 0.2% formaldehyde. [Figure 10] Graph showing the change in the amount of CVA16 VLP (native + denatured) (top row), native (middle row), and denatured (bottom row) per fixation period depending on the formaldehyde fixation temperature (room temperature, 37°C). [Figure 11]Graph showing the change in the amount of CVA16 VLP (native + denatured) (top row), native (middle row), and denatured (bottom row) at each fixation period using 0.01% to 0.05% glutaraldehyde. [Figure 12] Differential scanning calorimetry chart showing the temperature change of the main peak of VLPs in the presence of salt. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention relates to stabilized VLP vaccines used to prevent diseases caused by Enteroviruses, such as hand, foot and mouth disease, as well as methods for producing the same and stabilizing the VLP vaccines. In the present invention, a VLP vaccine is synonymous with a vaccine containing a virus-like particle (VLP).

[0013] In the present invention, the term "genus Enterovirus" refers to non-enveloped single-stranded RNA viruses of the genus Enterovirus in the family Picornaviridae. The genus Enterovirus is further classified into human enteroviruses and porcine enteroviruses, and human enteroviruses include poliovirus, Coxsackievirus A, Coxsackievirus B, echovirus, and other enteroviruses. Human enteroviruses are genotypically classified as human enteroviruses A to D. Human enterovirus A (HEV-A) includes Coxsackievirus A and the enterovirus group according to serotyping. Coxsackievirus A is further classified into serotypes 1 to 22 and 24, and the enterovirus group is serotypically classified into serotypes 68 to 71. Hand, foot, and mouth disease is primarily caused by serotypes EV71, Coxsackievirus A6, 10, and 16, which are genotypically classified as human enterovirus A. Therefore, in the present invention, the Enterovirus is preferably human enterovirus A (HEV-A), and more preferably serotypes EV71, and Coxsackievirus A6, 10, and 16. The genomic RNA of Enteroviruses contains a region encoding a viral structural protein called P1 and regions encoding viral nonstructural proteins called P2 and P3. P1 corresponds to the viral capsid, and this region is processed by the viral protease, a nonstructural protein, to cleave it into viral structural proteins VP0 to VP4. Enterovirus capsids can be formed from structural proteins VP0, VP1, and VP3 called procapsids, or from structural proteins VP1 to VP4 after VP0 is cleaved into VP2 and VP4. The amino acid sequences of VP0, VP1, VP2, VP3, and VP4 of the Enterovirus genus and the nucleotide sequences encoding them are known and readily available from databases such as the DNA Data Bank of Japan (DDBJ), EMBL-Bank / EBI, and GenBank / National Center for Biotechnology Information (NCBI). For example, the sequence information for EV71 BrCr is available under NCBI accession number U22521.

[0014] In the present invention, the term "derived from" means that the subject has immunological homology with the original virus or a certain homology with the amino acid sequence of the original virus, and is not limited to the production method. Immunological homology means that an immune response to the original virus is similarly elicited in the subject. The method for confirming immunological homology may be any means available to those skilled in the art and is not limited to a specific method. Generally, it can be confirmed by measuring whether antibodies induced by the subject to be expressed can neutralize the original virus, i.e., by measuring the neutralizing antibodies induced by the subject to be expressed. For example, the term "derived from the genus Enterovirus" means that an immune response to Enterovirus viruses is similarly elicited in the subject, or that the subject has a certain homology with the amino acid sequence of Enterovirus viruses, for example, a level of homology that is understood in microbiology as a virus of the genus Enterovirus. A certain degree of homology to an amino acid sequence typically means 80% or more, preferably 90% or more, for example, 95% or more, preferably 96% or more, more preferably 97% or more, even more preferably 98% or more, and optimally 99% or more homology. Any testing method commonly available to those skilled in the art can be used to test for homology, and for example, BLAST can be used with default parameters.

[0015] In the present invention, a "virus-like particle" refers to a hollow particle that has only a viral outer shell and does not contain a viral genome. By maintaining a particle structure similar to that of a virus, it is expected to have immunogenicity equivalent to or greater than that of an inactivated whole virus. In the present invention, the "particle structure" of a virus-like particle means a particle structure that is mainly composed of the outer shell of a virus, is similar to a virus, and is expected to have immunogenicity equivalent to or greater than that of an inactivated whole virus. In the present invention, "stabilized" means improved stability compared to vaccines containing VLPs obtained by known production methods. Specifically, it means improved thermal stability compared to unstabilized VLPs obtained by known production methods or vaccines containing such unstabilized VLPs. Here, "thermostable" refers to a relatively low rate of denaturation of VLPs or VLP vaccines after overnight incubation at 37°C. Vaccines containing such VLPs are useful as vaccines because they exhibit little denaturation due to body temperature even when administered to humans. More specifically, it refers to determining whether or not VLPs or VLP vaccines are denatured after overnight incubation at 37°C, and finding that at least 70%, preferably 80%, and more preferably 90% of the VLPs are intact. In the present invention, it is desirable for VLPs to be stable even at salt concentrations that can be administered in vivo, specifically, even at salt concentrations of 200 mM or less, preferably 154 mM or less. The stability of VLPs can be measured by methods such as absorbance measurement, differential scanning calorimetry (DSC), enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunofluorescence (IF). When enzyme-linked immunosorbent assay (ELISA) is used to measure the degree of denaturation of a VLP vaccine, antibodies that bind to native VLPs, antibodies that bind to denatured VLPs, or a combination of these, or antibodies that bind to VLPs regardless of their denatured state, can be measured. In the present invention, "at least 70%, 80%, or 90% native" means that the amount of antibodies that bind to native VLPs measured after incubation is 70%, 80%, or 90% or more compared to before incubation. In the present invention, stabilization can be achieved by incubating in a salt-containing buffer solution at 4° C. to 37° C. for 1 day to 3 weeks, preferably at 25° C. or below (room temperature) for 1 day to 3 weeks.

[0016] In the present invention, the term "salt" refers to a compound consisting of an anion and a cation. Salts used in the present invention include acidic, neutral, and basic normal salts. A normal salt refers to a salt that does not have an ionizable hydrogen atom and does not contain hydroxide ions or oxide ions as constituents. Preferred acidic normal salts include ammonium sulfate, magnesium chloride, and copper (II) sulfate. Preferred neutral normal salts include sodium chloride, potassium chloride, sodium nitrate, calcium chloride, and sodium sulfate. Preferred basic normal salts include sodium carbonate and potassium acetate. The salt is preferably contained in a buffer solution. More preferably, the salt is contained in the buffer solution at a concentration of 500 mM to 3 M. In the present invention, the term "buffer" refers to a solution with buffering properties, and is a mixture of a weak acid and its conjugate base, or a weak base and its conjugate acid. Specific examples include acetate buffer (acetic acid + sodium acetate), phosphate buffer (phosphoric acid + sodium phosphate), citrate buffer (citric acid + sodium citrate), citrate-phosphate buffer (citric acid + sodium phosphate), borate buffer, tartrate buffer, Tris buffer, phosphate-buffered saline, and HEPES (4-(2-hydroxyethyl)-1-piperazinesulfonic acid) buffer. A buffer that can be administered to humans is preferred, and phosphate buffer and citrate buffer are preferred. In the present invention, "immobilization" means chemically crosslinking stabilized virus-like particles, thereby enabling the retention of the particle structure of the virus-like particles that is resistant to heat denaturation. The retention of the particle structure can be confirmed by electron microscopy, sucrose density gradient centrifugation, dynamic light scattering, or detection methods using antibodies that specifically recognize VLPs (slot blot, Western blot, etc.). Specifically, formaldehyde is used for immobilization. Formaldehyde is an aldehyde represented by the chemical formula HCHO, and is also known as methylene oxide or methanal. In the present invention, the formaldehyde used for immobilization is preferably at a concentration of 0.01% to 0.2%. In the present invention, fixation is carried out using formaldehyde for one day or more, preferably one to three weeks. The treatment is carried out at a temperature between 4°C and 37°C, preferably at 25°C or below (room temperature). In addition, in the present invention, stabilization and fixation can also be carried out simultaneously. In this case, formaldehyde can be added to a buffer containing a salt used for stabilization, and treatment can be carried out simultaneously with the fixation. Furthermore, in the present invention, the fixation preferably includes a treatment of reducing the salt concentration after treatment with formaldehyde. The treatment of reducing the salt concentration can be performed by a conventional technique available for normal desalting, such as dilution with a salt-free or low-salt buffer, buffer exchange, reverse osmosis membrane method, or electrodialysis. By performing fixation with formaldehyde after stabilization or by simultaneously performing stabilization and fixation, the particle structure of the virus-like particles can be maintained even when the salt concentration is reduced to a level that allows administration to the body, thereby providing stabilized virus-like particles that are useful as vaccine antigens.

[0017] (VLP production) The polypeptides constituting the VLPs used in the present invention can be produced using the methods described in Patent Documents 1 or 2, or any of the methods for producing peptides, polypeptides, and proteins commonly available to those skilled in the art. Such methods include, but are not limited to, chemical synthesis and cellular expression systems. Those skilled in the art can easily select specific production methods and the reagents, conditions, and equipment used therein. For example, commercially available protein expression kits can be used according to the instructions provided with the kits. A preferred method for producing the polypeptides that constitute the VLPs used in the present invention is a method using a cell expression system. For methods using a cell expression system, there are no particular limitations on the expression vectors and cells that can be used. For example, commercially available vectors and cells can be used according to the instructions attached to the product. Such vectors include, but are not limited to, HaloTag vectors, pHEK293 vectors, BacPAK vectors, pRI101 DNA vectors, baculovirus vectors, and pcDNA vectors, and are selected appropriately depending on the cells used. Usable cells include, but are not limited to, CHO cells, HEK293 cells, HeLa cells, Vero cells, MDCK cells, E. coli cells, silkworm cells, and tobacco leaf cells. Since the VLPs used in the present invention are intended for administration to humans, they are preferably produced in an expression system using mammalian cells. While it is possible to produce such VLPs using expression systems using other cells, such as Escherichia coli, plant cells, and insect cells, mammalian cells are preferred from the viewpoint of post-translational modifications. Examples of mammalian cells suitable for producing the VLPs used in the present invention include CHO cells. Expression systems using such cells may be any means available to those skilled in the art, including, but not limited to, transient expression systems and stable expression systems using plasmid transfection. As an example, the VLPs used in the present invention can be produced using a CHO cell line according to the following steps: Extracting RNA encoding structural and / or nonstructural proteins from viruses or designing such RNA by artificial synthesis A process of synthesizing and amplifying DNA fragments from RNA using the RT-PCR method, and then incorporating the DNA fragments into an expression vector. A step of transfecting the obtained expression vector into cells. Optionally, selecting cells for stable expression. Those skilled in the art can easily select reagents, conditions, equipment, etc. for any step in the above production method. Furthermore, steps for confirming whether the intended results are being obtained can be appropriately inserted into each step. Examples of such confirmation steps include confirming the sequence of the extracted RNA using an appropriate sequencing method, such as cycle sequencing or pyrosequencing, or confirming that the target protein is contained in the obtained cell culture by Western blotting (WB) or the like. VLPs obtained by culturing CHO cell lines in this manner may be modified by phosphorylation, glycosylation, and the like. Modifications include, for example, post-translational modifications of CHO cells. Vaccines containing the stabilized VLPs of the present invention also contain polypeptides that have undergone such modifications. Because VLPs obtained from CHO cell lines have a structure identical to or similar to that of natural viral capsids, they can be used as active ingredients in vaccines.

[0018] (Stability evaluation) The stability of the stabilized VLPs of the present invention or vaccines containing such VLPs can be evaluated by measuring and comparing their antigen-binding activity under various conditions, for example, using absorbance measurements, differential scanning calorimetry (DSC), enzyme immunoassay (EIA), or enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and / or immunofluorescence (IF). In ELISA, a VLP-recognizing antibody is immobilized on a plate, the VLPs used in the present invention are added to the plate, and then a sample containing the desired antibody, such as a culture supernatant of antibody-producing cells or a purified antibody, is added. After washing, an enzyme substrate such as p-nitrophenyl phosphate is added to the plate, and the absorbance is measured to evaluate the antigen-binding activity of the sample. To evaluate the binding activity of an antibody, a peptide fragment, such as a C-terminal or N-terminal fragment, may be used as an antigen. Examples of VLP-recognizing antibodies include antibodies that specifically bind to native or denatured VLPs, either alone or in combination, or a combination of antibodies that bind to both native and denatured VLPs. Each antibody can be obtained by the following method. The inactivated whole particle CVA16 vaccine was mixed and emulsified with FCA (Freund's complete adjuvant) and immunized intraperitoneally in rats. Two weeks after the first immunization, the same antigen was mixed and emulsified with FICA (Freund's incomplete adjuvant) and immunized subcutaneously (second immunization). Two weeks after the second immunization, the same antigen was mixed and emulsified with FICA (Freund's incomplete adjuvant) and immunized subcutaneously with a predetermined amount (third immunization). Approximately two weeks after the third immunization, blood samples were taken and antibody activity was confirmed by ELISA. Spleens were collected from rats that showed an increase in antibody titer and were deemed ready for cell fusion, and hybridomas were produced to obtain antibodies. The obtained antibodies were selected by ELISA using virus particles, native VLPs, and denatured VLPs (heat-treated VLPs) as antigens. BIAcore (Pharmacia) may also be used to assess the activity of vaccines containing the stabilized VLPs of the present invention. In DSC, the stabilized VLP of the present invention exhibits a main peak Tm value increased by at least 3°C ​​or more, preferably 5°C or more, compared to a non-stabilized VLP.

[0019] (vaccines, vaccine preparations) The stabilized VLPs of the present invention can be formulated as vaccines, such as for hand, foot, and mouth disease. As used herein, the term "vaccine" refers to a formulation comprising the stabilized Enterovirus-derived VLPs of the present invention, as described above, or other Enterovirus-derived antigens, in a form that is capable of being administered to a vertebrate, and that induces an immune response sufficient to induce therapeutic immunity to alleviate infection and / or reduce at least one symptom of infection and / or enhance the effectiveness of another dose of VLP or antigen. As used herein, the term "immune response" refers to both humoral and cell-mediated immune responses. A humoral immune response involves, for example, stimulating the production of antibodies by B lymphocytes that neutralize infectious agents, prevent infectious agents from entering cells, prevent replication of the infectious agent, and / or protect host cells from infection and destruction. A cell-mediated immune response refers to an immune response exhibited by a vertebrate (e.g., a human) to an infectious agent that is mediated by T lymphocytes and / or other cells, such as macrophages, that prevents or mitigates the infection or reduces at least one symptom thereof. Vaccine formulations are generally described in Vaccine Design ("The subunit and adjuvant approach" (Powell MF and Newman MJ, eds.) (1995) Plenum Press, New York).

[0020] Compositions containing the stabilized VLPs of the present invention can be administered parenterally, for example, subcutaneously, intramuscularly, rectally, intranasally, intradermally, or intravenously, or orally. The dosage form is not particularly limited, and the compositions can be formulated into injections, emulsions, suppositories, patches, eye drops, nasal drops, capsules, tablets, granules, powders, syrups, and the like using conventional methods. In addition to the VLPs of the present invention, the vaccines of the present invention may contain other components such as buffers, isotonicity agents, soothing agents, preservatives, antioxidants, adjuvants, etc., depending on the purpose and use. Examples of buffering agents include buffer solutions such as phosphate, acetate, carbonate, and citrate. Examples of isotonic agents include sodium chloride, glycerin, and D-mannitol. Examples of soothing agents include benzyl alcohol. Examples of preservatives include thimerosal, parahydroxybenzoic acid esters, phenoxyethanol, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, antibiotics, and synthetic antibacterial agents. Examples of antioxidants include sulfites and ascorbic acid.

[0021] Examples of adjuvants include animal oils (e.g., squalene) or their hydrogenated oils; vegetable oils (e.g., palm oil, castor oil) or their hydrogenated oils; oil-based adjuvants such as anhydrous mannitol oleate, liquid paraffin, polybutene, caprylic acid, oleic acid, and higher fatty acid esters; water-soluble adjuvants such as PCPP, saponin, manganese gluconate, calcium gluconate, manganese glycerophosphate, soluble aluminum acetate, aluminum salicylate, acrylic acid copolymers, methacrylic acid copolymers, maleic anhydride copolymers, alkenyl derivative polymers, oil-in-water emulsions, and cationic lipids containing quaternary ammonium salts; precipitating adjuvants such as aluminum salts (e.g., aluminum hydroxide (alum), aluminum phosphate, and aluminum sulfate) or combinations thereof, and sodium hydroxide; microbial toxins such as cholera toxin and Escherichia coli heat-labile toxin; and other components (e.g., bentonite, muramyl dipeptide derivatives, and interleukins).

[0022] Pharmaceutical compositions suitable for injection use can be prepared by means known in the art. For example, sterile aqueous solutions (if water soluble) or dispersions, and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions can be used. In some embodiments, the composition may be sterile and may be fluid to facilitate injection. Pharmaceutically acceptable carriers include, for example, saline, buffered saline, dextrose, water, glycerol, isotonic aqueous buffer, and combinations thereof.

[0023] When a composition is intended for delivery to the respiratory (e.g., nasal) mucosa, it is typically formulated as an aqueous solution for administration as an aerosol or nasal drops, or as a dry powder for rapid deposition in the nasal passages, for example. Compositions for administration as nasal drops can contain one or more excipients of the type typically found in such compositions, such as preservatives, viscosity modifiers, tonicity agents, buffers, etc. Viscosity modifiers can be carboxyvinyl polymers, microcrystalline cellulose, chitosan, starch, polysaccharides, etc. Compositions for administration as dry powders can also contain one or more excipients typically found in such compositions, such as mucoadhesives, bulking agents, and agents for imparting appropriate powder flow and size characteristics. Bulking agents and powder flow and sizing agents can include mannitol, sucrose, trehalose, and xylitol.

[0024] In one embodiment, a vaccine comprising the stabilized Enterovirus VLPs of the invention may be formulated as a dry powder containing one or more Enterovirus VLPs as immunogens, an adjuvant such as MPL®, a biopolymer such as chitosan to promote adhesion to mucosal surfaces, and bulking agents such as mannitol and sucrose. For example, an Enterovirus vaccine may be formulated as 10 mg of a dry powder containing one or more Enterovirus VLPs, an MPL® adjuvant, chitosan mucoadhesive, and mannitol and sucrose as bulking agents and to provide suitable flow characteristics. The formulation can include about 7.0 mg chitosan (range 25 to 90% w / w), about 1.5 mg mannitol (range 0 to 50% w / w), about 1.5 mg sucrose (range 0 to 50% w / w), about 25 μg MPL® (range 0.1 to 5% w / w), and about 100 μg VLPs from the Enterovirus genus (range 0.05 to 5% w / w).

[0025] The Enterovirus-derived VLPs can be present at a concentration of about 0.01% (w / w) to about 80% (w / w). In one embodiment, the Enterovirus-derived VLPs can be formulated at a dosage of about 5 μg, about 15 μg, and about 50 μg (0.025, 0.075, and 0.25% w / w) per 10 mg of dry powder formulation for administration into both nostrils, or at a dosage of about 10 μg, about 30 μg, and about 100 μg (0.1, 0.3, and 1.0% w / w) for administration into one nostril. The formulation can be administered to one or both nostrils during each administration. There can also be a booster administration 1 to 12 weeks after the first administration to enhance the immune response. The content of Enterovirus VLPs in vaccines and vaccine formulations may range from 1 μg to 100 mg, preferably 1-500 μg, more preferably 5-200 μg, and most typically 1-100 μg. Each dose of Enterovirus VLPs is about 10 μg, about 30 μg, or about 100 μg in a total of 20 mg of dry powder when administered to both nostrils, or 10 mg of dry powder when administered to one nostril. The dry powder is characterized by less than 10% of the particles being less than 10 μm in diameter. The average particle size ranges from 10 to 500 μm in diameter.

[0026] In another embodiment, the VLPs and vaccine compositions can be formulated as liquids for subsequent administration to a subject. Liquid formulations intended for intranasal administration include VLPs of an Enterovirus genotype, an adjuvant, and a delivery agent such as chitosan. Liquid formulations for intramuscular, subcutaneous, or oral administration include VLPs of an Enterovirus genotype, an adjuvant, and a buffer, but do not include a delivery agent (e.g., chitosan).

[0027] The VLP and vaccine compositions described hereinabove can be lyophilized and stored anhydrous before being ready for use, at which point they are reconstituted with a diluent if used in a liquid formulation. Alternatively, the various components of the composition may be stored separately in a kit or device (any or all components are lyophilized). The components may remain in lyophilized form for dry formulations, or may be reconstituted for liquid formulations, and are mixed before use or administered separately to a patient. For administration as a dry powder, the vaccine may be pre-loaded into an intranasal delivery device or a topical (e.g., transdermal) delivery patch and stored until use. Such delivery devices and associated packaging preferably protect their contents and ensure stability.

[0028] (Methods for stimulating immune responses) The amount of VLPs in a dose of the stabilized VLP or vaccine formulation of the present invention is selected to elicit a strong immune response without significant and / or adverse side effects. Such an amount will vary depending on the route of administration and the adjuvant used. Generally, the dose administered to a patient should be sufficient to produce a beneficial therapeutic response in the patient over an extended period of time, or sufficient to induce the production of antibodies specific for the VLP. Thus, a sufficient amount of the composition is administered to a patient to elicit an immune response against the specific VLP and / or to alleviate, reduce, or treat the symptoms and / or complications from a disease or infection. An amount sufficient to accomplish this is defined as a "therapeutically effective dose."

[0029] The dosage of the stabilized Enterovirus-derived VLPs of the present invention can be about 1 μg to 10 mg, preferably about 2-50 μg, of stabilized Enterovirus-derived VLPs per formulation. A typical immunization regimen using the stabilized VLP formulations of the present invention involves administering a formulation containing 1-100 μg of VLPs in a predetermined number of doses (e.g., 1-4 doses). The dosage is determined based on the immunological activity generated by the composition, the condition of the patient, and the weight or body surface area of ​​the patient being treated. It is also adjusted depending on the presence, nature, and severity of any adverse side effects that may accompany the administration of a particular composition to a particular patient.

[0030] The stabilized VLPs and vaccine formulations of the present invention may be administered via non-mucosal or mucosal routes. These administrations may include in vivo administration by parenteral injection (e.g., intravenous, subcutaneous, and intramuscular) or other traditional direct routes, such as buccal / sublingual, rectal, oral, nasal, topical (e.g., transdermal and ophthalmic), intravaginal, intrapulmonary, intraarterial, intraperitoneal, intraocular, or intranasal routes, or directly into specific tissues. Alternatively, vaccines comprising the stabilized VLPs of the present invention may be administered via any of a variety of routes, including oral, topical, subcutaneous, mucosal, intravenous, intramuscular, intranasal, sublingual, transdermal, subdermal, intradermal, and suppositories. Administration may be accomplished simply by direct administration using a patch, needle, catheter, or related device, at a single time point, or at multiple time points. In a preferred embodiment, the stabilized VLPs of the present invention or vaccine formulations thereof are administered subcutaneously or intramuscularly.

[0031] In a further aspect, the present invention provides a method for eliciting a mucosal IgA immune response and a systemic IgG immune response by administering to a patient subcutaneously, intramuscularly, or to a mucosal surface a VLP or vaccine composition comprising one or more Enterovirus-derived VLPs, at least one effective adjuvant, and / or at least one delivery agent.

[0032] The present invention also contemplates providing a means for administering intranasal formulations of stabilized Enterovirus VLPs and at least one adjuvant or at least one delivery agent. The administration device may take the form of, for example, an aerosol delivery system and may be configured to administer a single dose or multiple doses. Such devices deliver a metered amount of vaccine to the nasal passages. Other examples of suitable devices include, but are not limited to, droppers, swabs, aerosolizers, air inhalers (e.g., Valois Monopowder Nasal Administration Device, Bespak UniDose DP), nebulizers, and inhalers. The device can deliver the VLP or vaccine formulation by a passive method, requiring the subject to inhale the formulation into the nostrils. Alternatively, the device can actively deliver the formulation by pumping or spraying a dose into the nostrils. The VLP formulation or vaccine can be delivered into one or both nostrils by the device. Administration can involve two devices per subject (one device per nostril). The actual dose of active ingredient (stabilized Enterovirus-derived VLPs) is about 1-1000 μg. In a preferred embodiment, the VLP or vaccine formulation is administered to the nasal mucosa by rapid deposition within the nasal passages from a device containing the formulation held near or inserted within the passages.

[0033] The present invention also provides a method for producing antibodies against one or more Enterovirus VLPs, comprising administering to a subject a vaccine comprising the stabilized VLPs of the present invention described above. These antibodies can be isolated and purified by routine methods in the art. Isolated antibodies specific to Enterovirus VLPs can be used in the development of diagnostic immunological assays. These assays can be used to detect Enterovirus viruses in clinical samples and identify the specific virus causing the infection. Alternatively, the isolated antibodies can be administered to a subject suspected of having a viral infection with an Enterovirus virus to confer passive or short-term immunity.

[0034] As described above, symptoms such as hand, foot, and mouth disease can be treated by administering a vaccine formulation containing the stabilized VLPs of the present invention to a subject. Symptoms of hand, foot, and mouth disease are well known in the art and include the appearance of 2-3 mm vesicular rashes on the oral mucosa and extremities, such as the palms, soles, and dorsum of the feet. Furthermore, infection with hand, foot, and mouth disease can rarely result in central nervous system complications, such as meningitis, cerebellar ataxia, acute flaccid paralysis (AFP), and encephalitis, primarily in infants. In particular, infection caused by EV71 can result in central nervous system complications. The present invention encompasses methods for alleviating and / or reducing at least one symptom associated with hand, foot, and mouth disease, or for inducing an immune response in a subject previously affected by hand, foot, and mouth disease, by administering a vaccine formulation containing the stabilized VLPs of the present invention to a subject. The reduction in symptoms can be determined by subjective or objective assessment, primarily by parental or physician evaluation of skin symptoms, or by administering appropriate tests or measurements, including, for example, assessing quality of life, inhibiting progression of symptoms or complications of hand, foot, and mouth disease, or reducing severity, or by appropriate assays (e.g., antibody titers and / or T cell activation assays). Subjects include both animals and humans.

[0035] [Example]

[0036] The contents of all patents and references expressly cited herein are hereby incorporated by reference in their entirety. The present invention will be specifically explained below by way of examples, but the present invention should not be construed as being limited to these examples.

[0037] Example 1 Preparation of CVA16 VLP CVA16VLP was obtained according to the method described in Patent Document 2 cited above. 1. Generation of CHO-S cells expressing CVA16VLP RNA was extracted from CVA16 using the Roche High Pure Viral RNA Kit according to the accompanying instructions. The P1 and 3CD protease regions of the resulting RNA were amplified by RT-PCR using the TaKaRa PrimeScript II High Fidelity RT-PCR Kit according to the accompanying instructions. The resulting amplified DNA fragment was ligated into the expression vector pCHO 1.0 (Thermo). Invitrogen One Shot® TOP10 Chemically Competent E. coli cells were transformed with the prepared plasmid according to the accompanying instructions, and the E. coli were then seeded and grown in medium. A VLP expression plasmid was extracted from the resulting E. coli using the Invitrogen Plasmid DNA Midiprep kit according to the accompanying instructions. The VLP expression cassette contained the P1 and 3CD proteases of CV16, as well as the CMV / EF1 Hybrid and EF2 / CMV Hybrid promoters. The 3CD protease is driven by a CMV / EF1 hybrid promoter, and P1 is driven by an EF2 / CMV hybrid promoter. VLPs were produced using Thermo's ExpiCHO Expression System. Specifically, ExpiCHO-S cells were cultured in ExpiCHO Expression Medium until a predetermined cell density was reached. The cultured ExpiCHO-S cells were transfected with ExpiFectamine CHO Reagent diluted in OptiPRO SFM Complexation Medium and the VLP expression plasmid. 2. Isolation and Purification of CVA16 VLPs Transfected cells were cultured in ExpiCHO Expression Medium, supplemented with ExpiCHO Feed and ExpiFectamine CHO Enhancer, and further cultured. Eight days after transfection, the culture was stopped and harvested. The resulting culture medium was centrifuged to separate the cells from the supernatant. The supernatant was collected and filtered (0.22 μm). The resulting filtrate was concentrated by ultrafiltration using a GE Healthcare AKTA fluxes filter with a molecular weight cutoff of 500 kDa. A pellet was prepared using a Hitachi CP80wx ultracentrifuge. The pellet was resuspended overnight in PBS. The suspension was fractionated by sucrose density gradient centrifugation (10-40%) using the CP80wx ultracentrifuge, and each fraction was subjected to WB to identify the VLP fraction. The VP0 band indicated that P1 had been cleaved by protease. These results indicated that CVA16 VLPs were present in the culture supernatant and were structurally similar to the native virus. VLPs could be isolated and purified without disrupting the culture.

[0038] 3. Establishment of a CHO-S cell line stably expressing CVA16VLP CHO-S cells were cultured in CD Forti CHO Medium (Thermo) with a final concentration of 8 mM glutamine (Thermo) at 15 mL, 10 6 viable cells / mL, and cultured at 37°C, 8% CO2, and 130–150 rpm until ready for transfection. 18 μg of expression vector was added to 750 μL of OptiPROSFM to prepare a DNA solution. 24 μL of FreeStyleMax (Thermo) was added to 726 μL of OptiPROSFM to prepare a TF solution. The TF solution was slowly added to the DNA solution, mixed by inversion, and incubated at room temperature for 10 minutes. The resulting DNA-TF solution was added to the cultured CHO-S cells and cultured at 37°C, 8% CO2, and 130–150 rpm for 24–48 hours. The cells were collected by centrifugation and diluted to 5 × 10 in culture medium + Puromycin (Thermo) at a final concentration of 7.5 μg / mL + methotrexate (Sigma) at a final concentration of 50–200 nM. 5 The cells were cultured at 37°C and 8% CO2 for 7 days, and then counted. When the viability was 20% or higher, the cells were collected by centrifugation and diluted to 3 × 10 5 The cells were cultured at an initial density of 3 × 10 cells / mL, with the medium changed every 3 days, until the viability reached 80% or higher. 5 Culture was continued until viable cells / mL were reached. Selection was performed by gradually increasing the methotrexate concentration from 201 to 500 nM, 501 to 800 nM, and 801 to 1000 nM, until a viability of 80% or higher was achieved. After the final methotrexate concentration reached 1000 nM, the cells were subcultured twice to adapt to methotrexate-containing medium. Culture of the cells after acclimation was continued, and after one week, the culture supernatant was collected and subjected to WB to confirm VLP expression levels. For cell line pools in which VLP expression was confirmed, single-cell cloning was performed on 96-well plates using limiting dilution and cultured at 37°C and 8% CO2 in CD Forti CHO Medium (Thermo) + final concentration 6mM glutamine (Thermo). Single cells were confirmed under a microscope, and the culture was scaled up sequentially as confluence was reached. Upon reaching confluence, VLP expression levels were confirmed by WB, and clones with confirmed expression were selected as stable VLP-expressing strains. The resulting VLP-stably expressing strain was cultured for 10 days, and the culture medium was harvested and centrifuged (2,630 × g, 4°C, 30 min) to separate the cells from the supernatant. The supernatant was then collected and filtered (0.45 μm and 0.22 μm). The resulting filtrate was concentrated 50-fold using a GE Healthcare AKTA flux s with a molecular weight cutoff of 500 kDa, then ultracentrifuged (100,000 × g, 4°C, 4 h). The resulting pellet was resuspended in PBS overnight. The suspension was centrifuged (13,000 × g, 4°C, 10 min), and the supernatant was filtered (0.22 μm). The supernatant was then fractionated (10–40%) by sucrose density gradient centrifugation (100,000 × g, 4°C, 4 h) to obtain the desired VLPs.

[0039] Example 2: Change in VLP amount due to thermal denaturation (1) The VLPs of CVA16 obtained in Example 1 were heat-treated at 60°C, and the amounts of native and denatured VLPs were measured. The amount of VLP was measured by ELISA using the neutralizing antibody 140-2C, the VP1 N-terminus-recognizing antibody 32-8H, and the neutralizing antibodies 312-1E-9G and 52-6B. The neutralizing antibody 140-2C reacts strongly with native VLP (undenatured) but not with denatured VLP. The VP1 N-terminal-recognizing antibody 32-8H reacts strongly with denatured VLP but weakly with native VLP. The neutralizing antibodies 312-1E-9G and 52-6B react with VLP regardless of the denatured state. The antibodies described above were obtained as follows. 50 μg of inactivated whole particle CVA16 vaccine (maximum 100 μL (1.25 ml / kg)) was mixed with Freund's complete adjuvant (FCA) and emulsified, and rats were immunized intraperitoneally. Two weeks after the first immunization, the same antigen was mixed with Freund's incomplete adjuvant (FICA) and emulsified, and 50 μg (maximum 100 μL (1.25 ml / kg)) was administered subcutaneously (second immunization). Two weeks after the second immunization, the same antigen was mixed with Freund's incomplete adjuvant (FICA) and emulsified, and 50 μg (maximum 100 μL (1.25 ml / kg)) was administered subcutaneously (third immunization). Approximately two weeks after the third immunization, blood samples were collected and antibody activity was confirmed by ELISA. Spleens were collected from rats that showed an increase in antibody titer and were deemed ready for cell fusion. Hybridomas were produced and antibodies were isolated. The isolated antibodies were selected by ELISA using virus particles, native VLPs, and heat-treated VLPs as antigens. These 140-2C, 32-8H, and 312-1E-G were immobilized and reacted with VLPs or virus particles, and the denatured state of VLPs was detected by detecting with HRP-labeled 52-6B. For ELISA, PBS-T (Takara Bio) was used as the washing solution, EzBlock Chemi (Atto Corporation) was used for diluting and blocking samples and antibodies, and ELISA POD Substrate TMB Kit (Nacalai Tesque, Inc.) was used as the HRP substrate. The results are shown in Figure 1. As a result, it was found that heat treatment at 60°C reduced the amount of undenatured VLPs and increased the amount of denatured VLPs. (2) The CVA16 VLPs and CVA16 virus obtained in Example 1 were treated for 10 minutes at 25°C, 30°C, 37°C, 45°C, 50°C, 55°C, 60°C, and 70°C, and the amounts of native and denatured VLPs were measured by ELISA in the same manner as in (1). The results are shown in Figure 2. The virus was grown in RD-A cells, and the culture supernatant was collected and centrifuged to separate the cells from the supernatant. The VLPs were then pelleted by ultracentrifugation (97,000 × g / 4 h / 4°C). The VLP pellet was suspended in D-PBS(-) (Nacalai Tesque, Inc.) and subjected to sucrose density gradient centrifugation (10-40%) to obtain the viral particle fraction. The buffer of the fraction was replaced with D-PBS(-) using an Amicon Ultra 100K (Millipore). As a result, it was found that VLPs began to denature at temperatures above 37°C, and viruses began to denature at temperatures above 45°C (Fig. 2).

[0040] Example 3 Fixation with formaldehyde VLPs were added to D-PBS(-) (Nacalai Tesque, Inc.) containing formaldehyde at concentrations of 0.005%, 0.01%, and 0.02%, and then incubated at 4°C, 25°C, and 37°C for 1 day, 1 week, and 2 weeks to be immobilized. After incubation, formaldehyde was neutralized with NaHSO4 and the samples were frozen at -30°C until analysis. Each sample was thawed and diluted with ELISA blocking buffer (EzBlock Chemi / ATTO Corporation), and then ELISA was performed. The results are shown in Figure 3. The results showed that incubation at 4°C and 25°C was stable, with no increase in VLPs detected at 32-8H for up to two weeks. However, incubation at 37°C for more than one week resulted in an increase in the amount of denatured VLPs detected at 32-8H and a decrease in native VLPs detected at 140-2C, indicating VLP denaturation (Figure 3). In other words, it was found that VLPs were denatured at 37°C even when fixed with formaldehyde alone.

[0041] Example 4 Fixation with formaldehyde before heat treatment VLPs were added to D-PBS(-) containing formaldehyde at concentrations of 0.005%, 0.01%, and 0.02%, and then incubated at 4°C, 25°C, and 37°C for 1 day, 1 week, and 2 weeks to be immobilized. After incubation, the formaldehyde was neutralized with NaHSO4 and frozen at -30°C. Each sample was thawed, diluted with D-PBS(-), and then incubated overnight at 37°C for heat treatment. After dilution with ELISA blocking buffer (EzBlock1Chemi / ATTO Corporation), the denatured state was confirmed by ELISA. As a control, a sample that had not been heat-treated (stored at 4°C after thawing) was also used to confirm the denatured state by ELISA. The ELISA results are shown in Figure 4 (left). As a result, it was found that VLPs that were heat-treated at 37°C after immobilization were denatured regardless of the immobilization temperature (Figure 4 (left)). As a control, inactivated virus (CVA16 virus was inactivated by adding formaldehyde to the purified CVA16 solution at a concentration of 0.01 w / v% and then incubating at 37°C for one week) and VLPs not treated with formaldehyde were diluted with D-PBS(-) to the same concentration, incubated overnight at 37°C, and heat-treated. They were then diluted with ELISA blocking buffer (EzBlock1Chemi / ATTO Corporation) to confirm their denaturation state. As a control, a sample that had not been heat-treated (stored at 4°C) was also used to confirm its denaturation state by ELISA. The ELISA results are shown in Figure 4 (right). As a result, the inactivated virus was not denatured even after heat treatment at 37°C overnight, but VLPs that had not been fixed with formaldehyde were denatured (Figure 4 (right)).

[0042] Example 5 VLP stabilization with salt The stability of VLPs was examined in buffers containing various concentrations of NaCl. The solutions were prepared in 20 mM phosphate buffer (pH 7.4) with NaCl concentrations of 150 mM, 300 mM, 500 mM, and 1000 mM. Also, 20 mM HEPES buffer (pH 7.4) was prepared so that NaCl concentrations were 100 mM, 300 mM, 400 mM, 500 mM, and 1000 mM. VLPs were added to each buffer solution, incubated at 37°C for 48 hours, and then heat-treated. Samples were collected 2, 6, 24, and 48 hours after the start of incubation and frozen at -30°C until analysis. The VLPs before and after heat treatment were diluted with ELISA blocking buffer (EzBlock1Chemi / ATTO Corporation), and their denatured state was confirmed by ELISA. The results are shown in Figure 5. The results showed that VLPs denatured over time at a typical salt concentration (150 mM), but that denaturation was suppressed as the salt concentration increased (Figure 5). Similar experiments were also performed using MOPS and Tris-HCl, and similar results were obtained at high salt concentrations. This suggests that the type of buffer solution does not affect salt stabilization.

[0043] Example 6: Examination of types of salt The 20 mM phosphate buffer (pH 7.4) was prepared so that the NaCl concentrations were 3 M, 2 M, and 1 M, the KCl concentration was 1 M, and the ammonium sulfate concentration was 1 M. Sucrose was diluted with D-PBS(-) to prepare a 1 M sucrose solution. VLPs were added to each solution, and the mixture was incubated at 37°C for 24 hours and then heat-treated. Samples were collected 2, 4, and 24 hours after the start of incubation and stored frozen at -30°C until analysis. After thawing, each sample was diluted with ELISA blocking buffer (EzBlock1 Chemi / ATTO Corporation), and the denatured state of VLPs was confirmed by ELISA. The results are shown in Figure 6. As a result, VLPs were denatured when incubated in D-PBS(-). Furthermore, the amount of denatured VLPs increased after 24 hours in D-PBS(-) containing 1M sucrose, suggesting that VLPs were denatured. On the other hand, VLPs were stable even at 37°C in solutions containing other salts. Furthermore, VLPs were shown to be stable at higher concentrations of NaCl, 1M KCl, and ammonium sulfate (Figure 6). Similar experiments were also performed with ammonium chloride, demonstrating that VLPs were stable at higher concentrations of ammonium chloride.

[0044] Example 7 Fixation with formaldehyde in high salt concentrations (1) CVA16 VLPs were added to a solution containing 0.05% formaldehyde (FA) in 20 mM phosphate buffer (pH 7.4) containing 0.5 M NaCl, and the solution was incubated at 4°C for 3 weeks for immobilization. Three weeks after the start of incubation, the formaldehyde was neutralized with NaHSO4. D-PBS(-) was added to reduce the salt concentration to approximately 170 mM, and the sample was then incubated overnight at 37°C for heat treatment. After incubation, the sample was diluted with ELISA blocking buffer (EzBlock1 Chemi / ATTO Corporation), and the denatured state of VLPs in the heat-treated and unheat-treated samples was examined by ELISA. The results are shown in Figure 7. As a result, the immobilized VLPs remained in the native form at over 100% of their original form even after incubation at 37°C, whereas the amount of native VLPs in the non-immobilized VLPs after incubation was reduced by over 60% compared to their original form (Figure 7). In other words, the immobilized VLPs remained unchanged after incubation at 37°C, whereas the non-immobilized VLPs were denatured. This indicates that formaldehyde fixation in a buffer with a high salt concentration is effective in stabilizing VLPs.

[0045] Example 8 Optimization of formaldehyde (FA) fixation (1) VLPs were immobilized in 20 mM phosphate buffer (pH 7.4) containing 1 M NaCl at formaldehyde concentrations of 0.01%, 0.02%, or 0.05% at 4°C for 1, 2, or 3 weeks. The cells were harvested 1, 2, or 3 weeks after the start of fixation, and the formaldehyde was neutralized with NaHSO4. Each harvested sample was diluted with 20 mM phosphate buffer (pH 7.4) to a final salt concentration of 155 mM and then heat-treated overnight at 37°C. The samples were diluted with ELISA blocking buffer (EzBlock1Chem / ATTO Corporation), and the degree of VLP denaturation in the samples before and after heat treatment was detected by ELISA. The results are shown in Figure 8. As a result, treatment with 0.01% or 0.02% formaldehyde reduced the amount of intact VLPs (VLPs detected at 140-2C) compared to before incubation and increased the amount of denatured VLPs (VLPs detected at 32-8H). However, treatment with 0.05% formaldehyde reduced the amount of intact VLPs to more than 95% of the amount before incubation, and reduced the amount of denatured VLPs. In other words, denaturation was not suppressed even after 3 weeks with 0.01% or 0.02% formaldehyde, but denaturation was suppressed with 0.05% formaldehyde. Furthermore, no significant difference was observed between 1 and 3 weeks of incubation with 0.05% formaldehyde (Figure 8). (2) Formaldehyde concentrations were adjusted to 0.05%, 0.08%, 0.1%, and 0.2% using 20 mM phosphate buffer (pH 7.4) containing 1 M NaCl, and VLPs were added to fix the VLPs. Measurements were then performed under the same conditions as in (1). As a result, no significant changes were observed up to 0.1%, but at 0.2%, the ELISA values ​​tended to be lower than under other conditions (Figure 9), suggesting that excessive cross-linking may have destroyed the epitope. This suggests that the concentration of formaldehyde used should be less than 0.2%, which is the level at which the epitope is not destroyed. (3) Temperature conditions for VLP immobilization A 20 mM phosphate buffer (pH 7.4) was prepared so that the formaldehyde concentration was 0.05% and the salt concentration was 1 M NaCl, and VLPs were added thereto to carry out fixation by formaldehyde cross-linking. Fixation conditions were room temperature or 37°C for 12 days to 3 weeks. Samples were taken 12 days, 2 weeks, or 3 weeks after the start of fixation, and glutaraldehyde was neutralized with NaHSO4. Each collected sample was diluted with 20 mM phosphate buffer to a salt concentration of 155 mM and then heat-treated overnight at 37°C. The degree of VLP denaturation in the samples before and after heat treatment was detected by ELISA. The results are shown in Figure 10. It was thought that immobilization at 37°C was more likely to cause VLP denaturation than immobilization at room temperature. (4) Fixation with glutaraldehyde (GA) VLPs were added to 20 mM phosphate buffer (pH 7.4) containing 1 M NaCl to prepare glutaraldehyde solutions at concentrations of 0.01%, 0.02%, or 0.05%. Fixation was performed at 4°C for 1, 2, or 3 weeks. Samples were taken 1, 2, or 3 weeks after the start of fixation, and the glutaraldehyde was neutralized with NaHSO4. Each collected sample was diluted with 20 mM phosphate buffer to a salt concentration of 155 mM and then heat-treated overnight at 37°C. The degree of VLP denaturation in the samples before and after heat treatment was detected by ELISA. As a result, immobilization with GA did not suppress VLP denaturation regardless of the concentration or duration (Figure 11).

[0046] Example 9 Determining the stability of VLPs in the presence of salt by differential scanning calorimetry The stability of the VLPs was examined by differential scanning calorimetry. Sample: The VLPs obtained in Example 1 were dialyzed against D-PBS(-) or 20 mM phosphate buffer containing 1 M NaCl. A Slide-A-Lyzer (pore size: 20 kDa) was used for dialysis. The sample inside the dialysis membrane was collected, and the external dialysis solution was used as a reference. Instrument used: MicroCal PEAQ-DSC Automated (Malvern Panalytical) Measurement conditions Scan range: 20-100℃ Scan rate: 60℃ / hr Feedback mode: None ·Analysis conditions Baseline parameters: Line Tm acquisition method: Automatic acquisition using analysis software The results are shown in Figure 12. As a result, the Tm value of the main peak increased by about 6°C by adding NaCl to the buffer solution, which was thought to have increased the stability of the VLPs. [Industrial Applicability]

[0047] The VLP vaccine derived from the genus Enterovirus of the present invention has excellent safety and good stability, and is useful for preventing diseases caused by viruses of the genus Enterovirus, such as hand, foot, and mouth disease. Furthermore, the method for producing the vaccine of the present invention is an industrially useful method that can produce a stable vaccine by a fixation method involving formaldehyde fixation in the presence of a high salt concentration.

Claims

1. A vaccine comprising stabilized virus-like particles derived from the genus Enterovirus.

2. A vaccine comprising stabilized virus-like particles derived from the genus Enterovirus, the virus-like particles having a fixed particle structure.

3. A vaccine comprising stabilized virus-like particles derived from the genus Enterovirus, wherein the Tm value of the main peak of the virus-like particles in differential scanning calorimetry is at least 3°C ​​higher than that of unstabilized virus-like particles.

4. The vaccine according to any one of claims 1 to 3, wherein the Enterovirus is Enterovirus 71 and / or Coxsackievirus A.

5. 1. A method for producing a vaccine comprising stabilized virus-like particles derived from an Enterovirus, said method comprising treating said virus-like particles with formaldehyde in a buffer comprising a salt.

6. 1. A method for stabilizing virus-like particles derived from the genus Enterovirus, said method comprising treating said virus-like particles with formaldehyde in a buffer containing a salt.

7. The method according to claim 5 , further comprising a treatment to reduce the salt concentration.

8. The method according to any one of claims 5 to 7, wherein the salt is selected from acidic, neutral and basic normal salts.

9. The method according to any one of claims 5 to 7, wherein the buffer contains the salt at a concentration of 500 nM to 3 M.

10. 8. The method according to any one of claims 5 to 7, wherein the formaldehyde is at a concentration of 0.01% to 0.2%.

11. The method according to any one of claims 5 to 7, wherein the treatment is carried out for a period of from 1 week to 3 weeks.

12. 8. The method according to any one of claims 5 to 7, wherein the treatment is carried out at a temperature between 4°C and 37°C.

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